Drug conjugates and their use for treating cancer, an autoimmune disease or an infectious disease

ABSTRACT

Drug-Linker-Ligand Conjugates are disclosed in which a Drug is linked to a Ligand via a peptide-based Linker unit. In one embodiment, the Ligand is an Antibody. Drug-Linker compounds and Drug compounds are also disclosed. Methods for treating cancer, an autoimmune disease or an infectious disease using the compounds and compositions of the invention are also disclosed.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/408,646, filed Mar. 20, 2009, which is a continuation of U.S. patentapplication Ser. No. 10/522,911, filed Jul. 7, 2005, which was filedunder 35 U.S.C. §371 as a national stage application of InternationalApplication No. PCT/US2003/24209, filed Jul. 31, 2003; which furtherclaims the benefit under 35 U.S.C. §119(e) of U.S. Provisional PatentApplication No. 60/400,403, filed Jul. 31, 2002. The disclosures of eachof the foregoing applications are hereby incorporated herein byreference.

FIELD OF THE INVENTION

The present invention is directed to Drug-Linker-Ligand Conjugates andto Drug-Linker Compounds, to compositions comprising aDrug-Linker-Ligand Conjugate or a Drug-Linker Compound, and to methodsfor using the same to treat cancer, an autoimmune disease or aninfectious disease.

BACKGROUND OF THE INVENTION

Several short peptidic compounds have been isolated from natural sourcesand found to have biological activity. Analogs of these compounds havealso been prepared, and some were found to have biological activity. Forexample, Auristatin E (U.S. Pat. No. 5,635,483 to Pettit et al.) is asynthetic analogue of the marine natural product Dolastatin 10, an agentthat inhibits tubulin polymerization by binding to the same site ontubulin as the anticancer drug vincristine (G. R. Pettit, Prog. Chem.Org. Nat. Prod., 70: 1-79 (1997)). Dolastatin 10, auristatin PE, andauristatin E are linear peptides having four amino acids, three of whichare unique to the dolastatin class of compounds. Both dolastatin 10 andauristatin PE are presently being used in human clinical trials to treatcancer. The structural differences between dolastatin 10 and auristatinE reside in the C-terminal residue, in which the thiazolephenethyl aminegroup of dolastatin 10 is replaced by a norephedrine unit in auristatinE.

The following references disclose dolastatin and auristatin compoundsand analogs thereof, and their use for treating cancer:

International Publication No. WO 96/33212 A1 to Teikoku Hormone Mfg.Co., Ltd.;

International Publication No. WO 96/14856 A1 to Arizona Board ofRegents;

European Patent Publication No. EP 695757 A2 to Arizona Board ofRegents;

European Patent Publication No. EP 695758 A2 to Arizona Board ofRegents;

European Patent Publication No. EP 695759 A2 to Arizona Board ofRegents;

International Publication No. WO 95/09864 A1 to Teikoku Hormone Mfg.Co., Ltd.;

International Publication No. WO 93/03054 A1 to Teikoku Hormone Mfg.Co., Ltd.;

U.S. Pat. No. 6,323,315 B1 to Pettit et al.;

G. R. Pettit et al., Anti-Cancer Drug Des. 13(4): 243-277 (1998);

G. R. Pettit et al., Anti-Cancer Drug Des. 10(7): 529-544 (1995); and

K. Miyazaki et al., Chem. Pharm. Bull. 43(10), 1706-18 (1995).

Despite in vitro data for compounds of the dolastatin class and itsanalogs, significant general toxicities at doses required for achievinga therapeutic effect compromise their efficacy in clinical studies.Accordingly, there is a clear need in the art for dolastatin derivativeshaving significantly lower toxicity, yet useful therapeutic efficiency,compared to current dolastatin drug therapies.

The recitation of any reference in Section 2 of this application is notan admission that the reference is prior art to this application.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides compounds of generalFormula Ia:

-   -   and pharmaceutically acceptable salts and solvates thereof        wherein,    -   L- is a Ligand unit;    -   -A- is a Stretcher unit;    -   a is 0 or 1;    -   each —W— is independently an Amino Acid unit;    -   —Y— is a Spacer unit;    -   w is an integer ranging from 0 to 12;    -   y is 0, 1 or 2;    -   p ranges from 1 to about 20; and    -   -D is a Drug unit of the formula

-   -   wherein, independently at each location:    -   R² is selected from -hydrogen and —C₁-C₈ alkyl;    -   R³ is selected from -hydrogen, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,        —O—(C₁-C₈ alkyl), aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈        carbocycle), —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈        heterocycle);    -   R⁴ is selected from -hydrogen, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,        —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈        carbocycle), —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈        heterocycle) wherein R⁵ is selected from —H and -methyl; or R⁴        and R⁵ join, have the formula —(CR^(a)R^(b))_(n)— wherein R^(a)        and R^(b) are independently selected from —H, —C₁-C₈ alkyl and        —C₃-C₈ carbocycle and n is selected from 2, 3, 4, 5 and 6, and        form a ring with the carbon atom to which they are attached;    -   R⁶ is selected from —H and —C₁-C₈ alkyl;    -   R⁷ is selected from —H, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,        —O—(C₁-C₈ alkyl), aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈        carbocycle), —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈        heterocycle);    -   each R⁸ is independently selected from —H, —OH, —C₁-C₈ alkyl,        —C₃-C₈ carbocycle and —O—(C₁-C₈ alkyl);    -   R⁹ is selected from —H and —C₁-C₈ alkyl;    -   R¹⁰ is selected from

-   -   Z is —O—, —S—, —NH— or —N(R¹⁴)—;    -   R¹¹ is selected from —H, —OH, —NH₂, —NHR¹⁴, —N(R¹⁴)₂, —C₁-C₈        alkyl, —C₃-C₈ carbocycle, —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈        alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈ carbocycle), —C₃-C₈ heterocycle        and —C₁-C₈ alkyl-(C₃-C₈ heterocycle); or R¹¹ is an oxygen atom        which forms a carbonyl unit (C═O) with the carbon atom to which        it is attached and a hydrogen atom on this carbon atom is        replaced by one of the bonds in the (C═O) double bond;    -   each R¹² is independently selected from -aryl and —C₃-C₈        heterocycle;    -   R¹³ is selected from —H, —OH, —NH₂, —NHR¹⁴, —N(R¹⁴)₂, —C₁-C₈        alkyl, —C₃-C₈ carbocycle, —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈        alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈ carbocycle), C₃-C₈ heterocycle        and —C₁-C₈ alkyl-(C₃-C₈ heterocycle); and    -   each R¹⁴ is independently —H or —C₁-C₈ alkyl.

In another aspect, the present invention provides compounds of generalformula Ib:

-   -   and pharmaceutically acceptable salts and solvates thereof    -   wherein,    -   L- is a Ligand unit;    -   -A- is a Stretcher unit;    -   a is 0 or 1;    -   each —W— is independently an Amino Acid unit;    -   —Y— is a Spacer unit;    -   w is an integer ranging from 0 to 12;    -   y is 0, 1 or 2;    -   p ranges from 1 to about 20; and    -   -D is a Drug unit of the formula

-   -   wherein, independently at each location:    -   R¹ is selected from —H, —C₁-C₈ alkyl and —C₃-C₈ carbocycle; and        R² is selected from —H and —C₁-C₈ alkyl; or R¹ and R² join, have        the formula —(CR^(a)R^(b))_(n)— wherein R^(a) and R^(b) are        independently selected from —H, —C₁-C₈ alkyl and —C₃-C₈        carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a        ring with the nitrogen atom to which they are attached;    -   R³ is selected from —H, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,        —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈        carbocycle), —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈        heterocycle);    -   R⁴ is selected from —H, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,        —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈        carbocycle), —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈        heterocycle) wherein R⁵ is selected from —H and -methyl; or R⁴        and R⁵ join, have the formula —(CR^(a)R^(b))_(n)— wherein R^(a)        and R^(b) are independently selected from —H, —C₁-C₈ alkyl and        —C₃-C₈ carbocycle and n is selected from 2, 3, 4, 5 and 6, and        form a ring with the carbon atom to which they are attached;    -   R⁶ is selected from —H and —C₁-C₈ alkyl;    -   R⁷ is selected from —H, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,        —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈        carbocycle), —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈        heterocycle);    -   each R⁸ is independently selected from —H, —OH, —C₁-C₈ alkyl,        —C₃-C₈ carbocycle and —O—(C₁-C₈ alkyl);    -   R⁹ is selected from —H and —C₁-C₈ alkyl;    -   R¹⁰ is selected from

-   -   X is —O—, —S—, —NH— or —N(R¹⁴)—, where X is bonded to Y when y        is 1 or 2, or X is bonded to W when y is 0;    -   Z is —O—, —S—, —NH— or —N(R¹⁴)—;    -   R¹¹ is selected from —H, —OH, —NH₂, —NHR¹⁴, —N(R¹⁴)₂, —C₁-C₈        alkyl, —C₃-C₈ carbocycle, —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈        alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈ carbocycle), —C₃-C₈ heterocycle        and —C₁-C₈ alkyl-(C₃-C₈ heterocycle); or R¹¹ is an oxygen atom        which forms a carbonyl unit (C═O) with the carbon atom to which        it is attached and a hydrogen atom on this carbon atom is        replaced by one of the bonds in the (C═O) double bond;    -   each R¹² is independently selected from -aryl and —C₃-C₈        heterocycle;    -   R¹³ is selected from —H, —OH, —NH₂, —NHR¹⁴, —N(R¹⁴)₂, —C₁-C₈        alkyl, —C₃-C₈ carbocycle, —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈        alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈ carbocycle), C₃-C₈ heterocycle        and —C₁-C₈ alkyl-(C₃-C₈ heterocycle);    -   each R¹⁴ is independently —H or —C₁-C₈ alkyl; and    -   R¹⁵ is -arylene-, —C₃-C₈ carbocyclo- or —C₃-C₈ heterocyclo-.

In another aspect, the present invention provides compounds of generalformula Ic:

-   -   and pharmaceutically acceptable salts and solvates thereof        wherein,    -   L- is a Ligand unit;    -   -A- is a Stretcher unit;    -   a is 0 or 1;    -   each —W— is independently an Amino Acid unit;    -   w is an integer ranging from 0 to 12;    -   each n is independently 0 or 1;    -   p ranges from 1 to about 20; and    -   each -D is independently:    -   (a) a Drug unit of the formula:

-   -   wherein, independently at each location:    -   R¹ is selected from —H, —C₁-C₈ alkyl and —C₃-C₈ carbocycle; and        R² is selected from —H and —C₁-C₈ alkyl; or R¹ and R² join, have        the formula —(CR^(a)R^(b))_(n)— wherein R^(a) and R^(b) are        independently selected from —H, —C₁-C₈ alkyl and —C₃-C₈        carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a        ring with the nitrogen atom to which they are attached;    -   R³ is selected from —H, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,        —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈        carbocycle), —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈        heterocycle);    -   R⁴ is selected from —H, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,        —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈        carbocycle), —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈        heterocycle) wherein R⁵ is selected from —H and -methyl; or R⁴        and R⁵ join, have the formula —(CR^(a)R^(b))_(n)— wherein R^(a)        and R^(b) are independently selected from —H, —C₁-C₈ alkyl and        —C₃-C₈ carbocycle and n is selected from 2, 3, 4, 5 and 6, and        form a ring with the carbon atom to which they are attached;    -   R⁶ is selected from —H and —C₁-C₈ alkyl;    -   R⁷ is selected from —H, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,        —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈        carbocycle), —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈        heterocycle);    -   each R⁸ is independently selected from —H, —OH, —C₁-C₈ alkyl,        -C₃-C₈ carbocycle and —O—(C₁-C₈ alkyl);    -   R⁹ is selected from —H and —C₁-C₈ alkyl;    -   R¹⁰ is selected from

-   -   X is —O—, —S—, —NH— or —N(R¹⁴)—, where X is bonded to —C(O)—        when y is 1 or 2, or X is bonded to —CH₂— when n is 0;    -   Z is —O—, —S—, —NH— or —N(R¹⁴)—;    -   R¹¹ is selected from —H, —OH, —NH₂, —NHR¹⁴, —N(R¹⁴)₂, —C₁-C₈        alkyl, —C₃-C₈ carbocycle, —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈        alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈ carbocycle), —C₃-C₈ heterocycle        and —C₁-C₈ alkyl-(C₃-C₈ heterocycle); or R¹¹ is an oxygen atom        which forms a carbonyl unit (C═O) with the carbon atom to which        it is attached and a hydrogen atom on this carbon atom is        replaced by one of the bonds in the (C═O) double bond;    -   each R¹² is independently selected from -aryl and —C₃-C₈        heterocycle;    -   R¹³ is selected from —H, —OH, —NH₂, —NHR¹⁴, —N(R¹⁴)₂, —C₁-C₈        alkyl, —C₃-C₈ carbocycle, —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈        alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈ carbocycle), C₃-C₈ heterocycle        and —C₁-C₈ alkyl-(C₃-C₈ heterocycle);    -   each R¹⁴ is independently —H or —C₁-C₈ alkyl; and    -   R¹⁵ is -arylene-, —C₃-C₈ carbocyclo- or —C₃-C₈ heterocyclo-; or    -   (b) a Drug unit of the formula:

-   -   wherein, independently at each location:    -   R² is selected from —H and —C₁-C₈ alkyl;    -   R³ is selected from —H, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,        —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈        carbocycle), —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈        heterocycle);    -   R⁴ is selected from —H, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,        —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈        carbocycle), —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈        heterocycle) wherein R⁵ is selected from —H and -methyl; or R⁴        and R⁵ join, have the formula —(CR^(a)R^(b))_(n)— wherein R^(a)        and R^(b) are independently selected from —H, —C₁-C₈ alkyl and        —C₃-C₈ carbocycle and n is selected from 2, 3, 4, 5 and 6, and        form a ring with the carbon atom to which they are attached;    -   R⁶ is selected from —H and —C₁-C₈ alkyl;    -   R⁷ is selected from —H, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,        —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈        carbocycle), —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈        heterocycle);    -   each R⁸ is independently selected from —H, —OH, —C₁-C₈ alkyl,        —C₃-C₈ carbocycle and —O—(C₁-C₈ alkyl);    -   R⁹ is selected from —H and —C₁-C₈ alkyl;    -   R¹⁰ is selected from

-   -   Z is —O—, —S—, —NH— or —N(R¹⁴)—;    -   R¹¹ is selected from —H, —OH, —NH₂, —NHR¹⁴, —N(R¹⁴)₂, —C₁-C₈        alkyl, —C₃-C₈ carbocycle, —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈        alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈ carbocycle), —C₃-C₈ heterocycle        and —C₁-C₈ alkyl-(C₃-C₈ heterocycle); or R¹¹ is an oxygen atom        which forms a carbonyl unit (C═O) with the carbon atom to which        it is attached and a hydrogen atom on this carbon atom is        replaced by one of the bonds in the (C═O) double bond;    -   each R¹² is independently selected from -aryl and —C₃-C₈        heterocycle;    -   R¹³ is selected from —H, —OH, —NH₂, —NHR¹⁴, —N(R¹⁴)₂, —C₁-C₈        alkyl, —C₃-C₈ carbocycle, —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈        alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈ carbocycle), C₃-C₈ heterocycle        and —C₁-C₈ alkyl-(C₃-C₈ heterocycle); and    -   each R¹⁴ is independently —H or —C₁-C₈ alkyl.

A compound of formula Ia, formula Ib, formula Ic or a pharmaceuticallyacceptable salt or solvate thereof (a “Drug-Linker-Ligand Conjugate”) isuseful for treating or preventing cancer, an autoimmune disease or aninfectious disease in an animal.

In another aspect, the present invention provides compounds of theformula IIa:

-   -   and pharmaceutically acceptable salts and solvates thereof    -   wherein, independently at each location:    -   R¹ is selected from —H, —C₁-C₈ alkyl and —C₃-C₈ carbocycle; and        R² is selected from —H and —C₁-C₈ alkyl; or R¹ and R² join, have        the formula —(CR^(a)R^(b))_(n)— wherein R^(a) and R^(b) are        independently selected from —H, —C₁-C₈ alkyl and —C₃-C₈        carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a        ring with the nitrogen atom to which they are attached;    -   R³ is selected from —H, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,        —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈        carbocycle), —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈        heterocycle);    -   R⁴ is selected from —H, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,        —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈        carbocycle), —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈        heterocycle) wherein R⁵ is selected from —H and -methyl; or R⁴        and R⁵ join, have the formula —(CR^(a)R^(b))_(n)— wherein R^(a)        and R^(b) are independently selected from —H, —C₁-C₈ alkyl and        —C₃-C₈ carbocycle and n is selected from 2, 3, 4, 5 and 6, and        form a ring with the carbon atom to which they are attached;    -   R⁶ is selected from —H and —C₁-C₈ alkyl;    -   R⁷ is selected from —H, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,        —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈        carbocycle), —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈        heterocycle);    -   each R⁸ is independently selected from —H, —OH, —C₁-C₈ alkyl,        —C₃-C₈ carbocycle and —O—(C₁-C₈ alkyl);    -   R⁹ is selected from —H and —C₁-C₈ alkyl;    -   X is —O—, —S—, —NH— or —N(R¹⁴)—;    -   R¹¹ is selected from —H, —OH, —NH₂, —NHR¹⁴, —N(R¹⁴)₂, —C₁-C₈        alkyl, —C₃-C₈ carbocycle, —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈        alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈ carbocycle), —C₃-C₈ heterocycle        and —C₁-C₈ alkyl-(C₃-C₈ heterocycle); or R¹¹ is an oxygen atom        which forms a carbonyl unit (C═O) with the carbon atom to which        it is attached and a hydrogen atom on this carbon atom is        replaced by one of the bonds in the (C═O) double bond;    -   each R¹² is independently selected from -aryl and —C₃-C₈        heterocycle;    -   each R¹⁴ is independently —H or —C₁-C₈ alkyl; and    -   R¹⁶ is —Yy-Ww-A′        wherein    -   each —W— is independently an Amino Acid unit;    -   —Y— is a Spacer unit;    -   w is an integer ranging from 0 to 12;    -   y is 0, 1 or 2; and    -   -A′ is selected from

wherein

-   -   G is selected from —Cl, —Br, —I, —O-mesyl and —O-tosyl;    -   J is selected from —Cl, —Br, —I, —F, —OH, —O—N-succinimide,        —O-(4-nitrophenyl), —O-pentafluorophenyl, —O-tetrafluorophenyl        and —O—C(O)—OR¹⁸;    -   R¹⁷ is selected from —C₁-C₁₀ alkylene-, —C₃-C₈ carbocyclo-,        —O—(C₁-C₈ alkyl)-, -arylene-, —C₁-C₁₀ alkylene-arylene-,        -arylene-C₁-C₁₀ alkylene-, —C₁-C₁₀ alkylene-(C₃-C₈ carbocyclo)-,        —(C₃-C₈ carbocyclo)-C₁-C₁₀ alkylene-, —C₃-C₈ heterocyclo-,        —C₁-C₁₀ alkylene-(C₃-C₈ heterocyclo)-, —(C₃-C₈        heterocyclo)-C₁-C₁₀ alkylene-, —(CH₂CH₂O)_(r)—, and        —(CH₂CH₂O)_(r)—CH₂—; r is an integer ranging from 1-10; and    -   R¹⁸ is —C₁-C₈ alkyl or -aryl.

In another aspect, the present invention provides compounds of theformula Ib:

-   -   and pharmaceutically acceptable salts and solvates thereof    -   wherein, independently at each location:    -   R¹ is selected from —H, —C₁-C₈ alkyl and —C₃-C₈ carbocycle; and        R² is selected from —H and —C₁-C₈ alkyl; or R¹ and R² join, have        the formula —(CR^(a)R^(b))_(n)— wherein R^(a) and R^(b) are        independently selected from —H, —C₁-C₈ alkyl and —C₃-C₈        carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a        ring with the nitrogen atom to which they are attached;    -   R³ is selected from —H, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,        —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈        carbocycle), —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈        heterocycle);    -   R⁴ is selected from —H, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,        —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈        carbocycle), —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈        heterocycle) wherein R⁵ is selected from —H and -methyl; or R⁴        and R⁵ join, have the formula —(CR^(a)R^(b))_(n)— wherein R^(a)        and R^(b) are independently selected from —H, —C₁-C₈ alkyl and        —C₃-C₈ carbocycle and n is selected from 2, 3, 4, 5 and 6, and        form a ring with the carbon atom to which they are attached;    -   R⁶ is selected from —H and —C₁-C₈ alkyl;    -   R⁷ is selected from —H, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,        —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈        carbocycle), —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈        heterocycle);    -   each R⁸ is independently selected from —H, —OH, —C₁-C₈ alkyl,        —C₃-C₈ carbocycle and —O—(C₁-C₈ alkyl);    -   R⁹ is selected from —H and —C₁-C₈ alkyl;    -   X is —O—, —S—, —NH— or —N(R¹⁴)—;    -   R¹¹ is selected from —H, —OH, —NH₂, —NHR¹⁴, —N(R¹⁴)₂, —C₁-C₈        alkyl, —C₃-C₈ carbocycle, —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈        alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈ carbocycle), —C₃-C₈ heterocycle        and —C₁-C₈ alkyl-(C₃-C₈ heterocycle); or R¹¹ is an oxygen atom        which forms a carbonyl unit (C═O) with the carbon atom to which        it is attached and a hydrogen atom on this carbon atom is        replaced by one of the bonds in the (C═O) double bond;    -   R¹³ is selected from hydrogen, —OH, —NH₂, —NHR¹⁴, —N(R¹⁴)₂,        C₁-C₈ alkyl, C₃-C₈ carbocycle, —O—(C₁-C₈ alkyl), -aryl,        alkyl-aryl, alkyl-(C₃-C₈ carbocycle), C₃-C₈ heterocycle and        alkyl-(C₃-C₈ heterocycle);    -   each R¹⁴ is independently —H or —C₁-C₈ alkyl;    -   R¹⁵ is -arylene-, —C₃-C₈ carbocyclo- or —C₃-C₈ heterocyclo-; and    -   R¹⁶ is —Yy-Ww-A′        wherein    -   each —W— is independently an Amino Acid unit;    -   —Y— is a Spacer unit;    -   w is an integer ranging from 0 to 12;    -   y is 0, 1 or 2; and    -   -A′ is selected from

wherein

-   -   G is selected from —Cl, —Br, —I, —O-mesyl and —O-tosyl;    -   J is selected from —Cl, —Br, —I, —F, —OH, —O—N-succinimide,        —O-(4-nitrophenyl), —O-pentafluorophenyl, —O-tetrafluorophenyl        and —O—C(O)—OR¹⁸;    -   R¹⁷ is selected from —C₁-C₁₀ alkylene-, —C₃-C₈ carbocyclo-,        —O—(C₁-C₈ alkyl)-, -arylene-, —C₁-C₁₀ alkylene-arylene-,        -arylene-C₁-C₁₀ alkylene-, —C₁-C₁₀ alkylene-(C₃-C₈ carbocyclo)-,        —(C₃-C₈ carbocyclo)-C₁-C₁₀ alkylene-, —C₃-C₈ heterocyclo-,        —C₁-C₁₀ alkylene-(C₃-C₈ heterocyclo)-, —(C₃-C₈        heterocyclo)-C₁-C₁₀alkylene-, —(CH₂CH₂O)_(r)—, and        —(CH₂CH₂O)_(r)—CH₂—; r is an integer ranging from 1-10; and    -   R¹⁸ is —C₁-C₈ alkyl or -aryl.

In another aspect, the present invention provides compounds of theformula IIc:

-   -   and pharmaceutically acceptable salts and solvates thereof    -   wherein, independently at each location:    -   R¹ is selected from —H, —C₁-C₈ alkyl and —C₃-C₈ carbocycle; and        R² is selected from —H and —C₁-C₈ alkyl; or R¹ and R² join, have        the formula —(CR^(a)R^(b))_(n)— wherein R^(a) and R^(b) are        independently selected from —H, —C₁-C₈ alkyl and —C₃-C₈        carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a        ring with the nitrogen atom to which they are attached;    -   R³ is selected from —H, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,        —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈        carbocycle), —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈        heterocycle);    -   R⁴ is selected from —H, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,        —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈        carbocycle), —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈        heterocycle) wherein R⁵ is selected from —H and -methyl; or R⁴        and R⁵ join, have the formula —(CR^(a)R^(b))_(n)— wherein R^(a)        and R^(b) are independently selected from —H, —C₁-C₈ alkyl and        —C₃-C₈ carbocycle and n is selected from 2, 3, 4, 5 and 6, and        form a ring with the carbon atom to which they are attached;    -   R⁶ is selected from —H and —C₁-C₈ alkyl;    -   R⁷ is selected from —H, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,        —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈        carbocycle), —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈        heterocycle);    -   each R⁸ is independently selected from —H, —OH, —C₁-C₈ alkyl,        —C₃-C₈ carbocycle and —O—(C₁-C₈ alkyl);    -   R⁹ is selected from —H and —C₁-C₈ alkyl;    -   X is —O—, —S—, —NH— or —N(R¹⁴)—;    -   R¹¹ is selected from —H, —OH, —NH₂, —NHR¹⁴, —N(R¹⁴)₂, —C₁-C₈        alkyl, —C₃-C₈ carbocycle, —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈        alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈ carbocycle), —C₃-C₈ heterocycle        and —C₁-C₈ alkyl-(C₃-C₈ heterocycle); or R¹¹ is an oxygen atom        which forms a carbonyl unit (C═O) with the carbon atom to which        it is attached and a hydrogen atom on this carbon atom is        replaced by one of the bonds in the (C═O) double bond;    -   each R¹² is independently selected from -aryl and —C₃-C₈        heterocycle;    -   each R¹⁴ is independently —H or —C₁-C₈ alkyl;    -   R¹⁶ is —Yy-Ww-A′        wherein    -   each —W— is independently an Amino Acid unit;    -   —Y— is a Spacer unit;    -   w is an integer ranging from 0 to 12;    -   y is 0, 1 or 2; and    -   -A′ is selected from

wherein

-   -   G is selected from —Cl, —Br, —I, —O-mesyl and —O-tosyl;    -   J is selected from —Cl, —Br, —I, —F, —OH, —O—N-succinimide,        —O-(4-nitrophenyl), —O-pentafluorophenyl, —O-tetrafluorophenyl        and —O—C(O)—OR¹⁸;    -   R¹⁷ is selected from —C₁-C₁₀ alkylene-, —C₃-C₈ carbocyclo-,        —O—(C₁-C₈ alkyl)-, -arylene-, —C₁-C₁₀ alkylene-arylene-,        -arylene-C₁-C₁₀ alkylene-, —C₁-C₁₀ alkylene-(C₃-C₈ carbocyclo)-,        —(C₃-C₈ carbocyclo)-C₁-C₁₀ alkylene-, —C₃-C₈ heterocyclo-,        —C₁-C₁₀ alkylene-(C₃-C₈ heterocyclo)-, —(C₃-C₈        heterocyclo)-C₁-C₁₀ alkylene-, —(CH₂CH₂O)_(r)—, and        —(CH₂CH₂O)_(r)—CH₂—; r is an integer ranging from 1-10; and    -   R¹⁸ is —C₁-C₈ alkyl or -aryl.

In another aspect, the present invention provides compounds of theformula IId:

-   -   and pharmaceutically acceptable salts and solvates thereof    -   wherein, independently at each location:    -   R¹ is selected from —H, —C₁-C₈ alkyl and —C₃-C₈ carbocycle; and        R² is selected from —H and —C₁-C₈ alkyl; or R¹ and R² join, have        the formula —(CR^(a)R^(b))_(n)— wherein R^(a) and R^(b) are        independently selected from —H, —C₁-C₈ alkyl and —C₃-C₈        carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a        ring with the nitrogen atom to which they are attached;    -   R³ is selected from —H, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,        —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈        carbocycle), —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈        heterocycle);    -   R⁴ is selected from —H, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,        —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈        carbocycle), —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈        heterocycle) wherein R⁵ is selected from —H and -methyl; or R⁴        and R⁵ join, have the formula —(CR^(a)R^(b))_(n)— wherein R^(a)        and R^(b) are independently selected from —H, —C₁-C₈ alkyl and        —C₃-C₈ carbocycle and n is selected from 2, 3, 4, 5 and 6, and        form a ring with the carbon atom to which they are attached;    -   R⁶ is selected from —H and —C₁-C₈ alkyl;    -   R⁷ is selected from —H, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,        —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈        carbocycle), —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈        heterocycle);    -   each R⁸ is independently selected from —H, —OH, —C₁-C₈ alkyl,        —C₃-C₈ carbocycle and —O—(C₁-C₈ alkyl);    -   R⁹ is selected from —H and —C₁-C₈ alkyl;    -   X is —O—, —S—, —NH— or —N(R¹⁴)—;    -   R¹¹ is selected from —H, —OH, —NH₂, —NHR¹⁴, —N(R¹⁴)₂, —C₁-C₈        alkyl, —C₃-C₈ carbocycle, —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈        alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈ carbocycle), —C₃-C₈ heterocycle        and —C₁-C₈ alkyl-(C₃-C₈ heterocycle); or R¹¹ is an oxygen atom        which forms a carbonyl unit (C═O) with the carbon atom to which        it is attached and a hydrogen atom on this carbon atom is        replaced by one of the bonds in the (C═O) double bond;    -   each R¹² is independently selected from -aryl and —C₃-C₈        heterocycle;    -   each R¹⁴ is independently —H or —C₁-C₈ alkyl;    -   R¹⁵ is -arylene-, —C₃-C₈ carbocyclo- or —C₃-C₈ heterocyclo-;    -   R¹⁶ is —Yy-Ww-A′        wherein    -   each —W— is independently an Amino Acid unit;    -   —Y— is a Spacer unit;    -   w is an integer ranging from 0 to 12;    -   y is 0, 1 or 2; and    -   -A′ is selected from

wherein

-   -   G is selected from —Cl, —Br, —I, —O-mesyl and —O-tosyl;    -   J is selected from —Cl, —Br, —I, —F, —OH, —O—N-succinimide,        —O-(4-nitrophenyl), —O-pentafluorophenyl, —O-tetrafluorophenyl        and —O—C(O)—OR¹⁸;    -   R¹⁷ is selected from —C₁-C₁₀ alkylene-, —C₃-C₈ carbocyclo-,        —O—(C₁-C₈ alkyl)-, -arylene-, —C₁-C₁₀ alkylene-arylene-,        -arylene-C₁-C₁₀ alkylene-, —C₁-C₁₀ alkylene-(C₃-C₈ carbocyclo)-,        —(C₃-C₈ carbocyclo)-C₁-C₁₀ alkylene-, —C₃-C₈ heterocyclo-,        —C₁-C₁₀ alkylene-(C₃-C₈ heterocyclo)-, —(C₃-C₈        heterocyclo)-C₁-C₁₀alkylene-, —(CH₂CH₂O)_(r)—, and        —(CH₂CH₂O)_(r)—CH₂—; r is an integer ranging from 1-10; and    -   R¹⁸ is —C₁-C₈ alkyl or -aryl

In another aspect, the present invention provides compounds of theformula IIe:

-   -   and pharmaceutically acceptable salts and solvates thereof    -   wherein, independently at each location:    -   R¹ is selected from —H, —C₁-C₈ alkyl and —C₃-C₈ carbocycle; and        R² is selected from —H and —C₁-C₈ alkyl; or R¹ and R² join, have        the formula —(CR^(a)R^(b))_(n)— wherein R^(a) and R^(b) are        independently selected from —H, —C₁-C₈ alkyl and —C₃-C₈        carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a        ring with the nitrogen atom to which they are attached;    -   R³ is selected from —H, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,        —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈        carbocycle), —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈        heterocycle);    -   R⁴ is selected from —H, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,        —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈        carbocycle), —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈        heterocycle) wherein R⁵ is selected from —H and -methyl; or R⁴        and R⁵ join, have the formula —(CR^(a)R^(b))_(n)— wherein R^(a)        and R^(b) are independently selected from —H, —C₁-C₈ alkyl and        —C₃-C₈ carbocycle and n is selected from 2, 3, 4, 5 and 6, and        form a ring with the carbon atom to which they are attached;    -   R⁶ is selected from —H and —C₁-C₈ alkyl;    -   R⁷ is selected from —H, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,        —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈        carbocycle), —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈        heterocycle);    -   each R⁸ is independently selected from —H, —OH, —C₁-C₈ alkyl,        —C₃-C₈ carbocycle and —O—(C₁-C₈ alkyl);    -   R⁹ is selected from —H and —C₁-C₈ alkyl;    -   X is —O—, —S—, —NH— or —N(R¹⁴)—;    -   Z is —O—, —S—, —NH— or —N(R¹⁴)—;    -   R¹¹ is selected from —H, —OH, —NH₂, —NHR¹⁴, —N(R¹⁴)₂, —C₁-C₈        alkyl, —C₃-C₈ carbocycle, —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈        alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈ carbocycle), —C₃-C₈ heterocycle        and —C₁-C₈ alkyl-(C₃-C₈ heterocycle); or R¹¹ is an oxygen atom        which forms a carbonyl unit (C═O) with the carbon atom to which        it is attached and a hydrogen atom on this carbon atom is        replaced by one of the bonds in the (C═O) double bond;    -   each R¹² is independently selected from -aryl and —C₃-C₈        heterocycle;    -   each R¹⁴ is independently —H or —C₁-C₈ alkyl;    -   R¹⁵ is -arylene-, —C₃-C₈ carbocyclo- or —C₃-C₈ heterocyclo-;    -   R¹⁶ is —Yy-Ww-A′        wherein    -   each —W— is independently an Amino Acid unit;    -   —Y— is a Spacer unit;    -   w is an integer ranging from 0 to 12;    -   y is 0, 1 or 2; and    -   -A′ is selected from

wherein

-   -   G is selected from —Cl, —Br, —I, —O-mesyl and —O-tosyl;    -   J is selected from —Cl, —Br, —I, —F, —OH, —O—N-succinimide,        —O-(4-nitrophenyl), —O-pentafluorophenyl, —O-tetrafluorophenyl        and —O—C(O)—OR¹⁸;    -   R¹⁷ is selected from —C₁-C₁₀ alkylene-, —C₃-C₈ carbocyclo-,        —O—(C₁-C₈ alkyl)-, -arylene-, —C₁-C₁₀ alkylene-arylene-,        -arylene-C₁-C₁₀ alkylene-, —C₁-C₁₀ alkylene-(C₃-C₈ carbocyclo)-,        —(C₃-C₈ carbocyclo)-C₁-C₁₀ alkylene-, —C₃-C₈ heterocyclo-,        —C₁-C₁₀ alkylene-(C₃-C₈ heterocyclo)-, —(C₃-C₈        heterocyclo)-C₁-C₁₀alkylene-, —(CH₂CH₂O)_(r)—, and        —(CH₂CH₂O)_(r)—CH₂—; r is an integer ranging from 1-10; and    -   R¹⁸ is —C₁-C₈ alkyl or -aryl.

In another aspect, the present invention provides compounds of theformula IIf:

-   -   and pharmaceutically acceptable salts and solvates thereof    -   wherein, independently at each location:    -   R¹ is selected from —H, —C₁-C₈ alkyl and —C₃-C₈ carbocycle; and        R² is selected from —H and —C₁-C₈ alkyl; or R¹ and R² join, have        the formula —(CR^(a)R^(b))_(n)— wherein R^(a) and R^(b) are        independently selected from —H, —C₁-C₈ alkyl and —C₃-C₈        carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a        ring with the nitrogen atom to which they are attached;    -   R³ is selected from —H, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,        —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈        carbocycle), —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈        heterocycle);    -   R⁴ is selected from —H, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,        —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈        carbocycle), —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈        heterocycle) wherein R⁵ is selected from —H and -methyl; or R⁴        and R⁵ join, have the formula —(CR^(a)R^(b))_(n)— wherein R^(a)        and R^(b) are independently selected from —H, —C₁-C₈ alkyl and        —C₃-C₈ carbocycle and n is selected from 2, 3, 4, 5 and 6, and        form a ring with the carbon atom to which they are attached;    -   R⁶ is selected from —H and —C₁-C₈ alkyl;    -   R⁷ is selected from —H, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,        —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈        carbocycle), —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈        heterocycle);    -   each R⁸ is independently selected from —H, —OH, —C₁-C₈ alkyl,        —C₃-C₈ carbocycle and —O—(C₁-C₈ alkyl);    -   R⁹ is selected from —H and —C₁-C₈ alkyl;    -   X is —O—, —S—, —NH— or —N(R¹⁴)—;    -   Z is —O—, —S—, —NH— or —N(R¹⁴)—;    -   R¹¹ is selected from —H, —OH, —NH₂, —NHR¹⁴, —N(R¹⁴)₂, —C₁-C₈        alkyl, —C₃-C₈ carbocycle, —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈        alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈ carbocycle), —C₃-C₈ heterocycle        and —C₁-C₈ alkyl-(C₃-C₈ heterocycle); or R¹¹ is an oxygen atom        which forms a carbonyl unit (C═O) with the carbon atom to which        it is attached and a hydrogen atom on this carbon atom is        replaced by one of the bonds in the (C═O) double bond;    -   each R¹² is independently selected from -aryl and —C₃-C₈        heterocycle;    -   each R¹⁴ is independently —H or —C₁-C₈ alkyl;    -   R¹⁵ is -arylene-, —C₃-C₈ carbocyclo- or —C₃-C₈ heterocyclo-;    -   R¹⁶ is —Yy-Ww-A′        wherein    -   each —W— is independently an Amino Acid unit;    -   —Y— is a Spacer unit;    -   w is an integer ranging from 0 to 12;    -   y is 0, 1 or 2; and    -   -A′ is selected from

wherein

-   -   G is selected from —Cl, —Br, —I, —O-mesyl and —O-tosyl;    -   J is selected from —Cl, —Br, —I, —F, —OH, —O—N-succinimide,        —O-(4-nitrophenyl), —O-pentafluorophenyl, —O-tetrafluorophenyl        and —O—C(O)—OR¹⁸;    -   R¹⁷ is selected from —C₁-C₁₀ alkylene-, —C₃-C₈ carbocyclo-,        —O—(C₁-C₈ alkyl)-, -arylene-, —C₁-C₁₀ alkylene-arylene-,        -arylene-C₁-C₁₀ alkylene-, —C₁-C₁₀ alkylene-(C₃-C₈ carbocyclo)-,        —(C₃-C₈ carbocyclo)-C₁-C₁₀ alkylene-, —C₃-C₈ heterocyclo-,        —C₁-C₁₀ alkylene-(C₃-C₈ heterocyclo)-, —(C₃-C₈        heterocyclo)-C₁-C₁₀alkylene-, —(CH₂CH₂O)_(r)—, and        —(CH₂CH₂O)_(r)—CH₂—; r is an integer ranging from 1-10; and    -   R¹⁸ is —C₁-C₈ alkyl or -aryl.

In another aspect, the present invention provides compounds of theformula IIg:

-   -   and pharmaceutically acceptable salts and solvates thereof    -   wherein, independently at each location:    -   R² is selected from —H and —C₁-C₈ alkyl;    -   R³ is selected from —H, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,        —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈        carbocycle), —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈        heterocycle);    -   R⁴ is selected from —H, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,        —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈        carbocycle), —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈        heterocycle) wherein R⁵ is selected from —H and -methyl; or R⁴        and R⁵ join, have the formula —(CR^(a)R^(b))_(n)— wherein R^(a)        and R^(b) are independently selected from —H, —C₁-C₈ alkyl and        —C₃-C₈ carbocycle and n is selected from 2, 3, 4, 5 and 6, and        form a ring with the carbon atom to which they are attached;    -   R⁶ is selected from —H and —C₁-C₈ alkyl;    -   R⁷ is selected from —H, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,        —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈        carbocycle), —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈        heterocycle);    -   each R⁸ is independently selected from —H, —OH, —C₁-C₈ alkyl,        —C₃-C₈ carbocycle and —O—(C₁-C₈ alkyl);    -   R⁹ is selected from —H and —C₁-C₈ alkyl;    -   Z is —O—, —S—, —NH— or —N(R¹⁴)—;    -   R¹¹ is selected from —H, —OH, —NH₂, —NHR¹⁴, —N(R¹⁴)₂, —C₁-C₈        alkyl, —C₃-C₈ carbocycle, —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈        alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈ carbocycle), —C₃-C₈ heterocycle        and —C₁-C₈ alkyl-(C₃-C₈ heterocycle); or R¹¹ is an oxygen atom        which forms a carbonyl unit (C═O) with the carbon atom to which        it is attached and a hydrogen atom on this carbon atom is        replaced by one of the bonds in the (C═O) double bond;    -   each R¹² is independently selected from -aryl and —C₃-C₈        heterocycle;    -   each R¹⁴ is independently —H or —C₁-C₈ alkyl;    -   R¹⁶ is —Yy-Ww-A′        wherein    -   each —W— is independently an Amino Acid unit;    -   —Y— is a Spacer unit;    -   w is an integer ranging from 0 to 12;    -   y is 0, 1 or 2; and    -   -A′ is selected from

wherein

-   -   G is selected from —Cl, —Br, —I, —O-mesyl and —O-tosyl;    -   J is selected from —Cl, —Br, —I, —F, —OH, —O—N-succinimide,        —O-(4-nitrophenyl), —O-pentafluorophenyl, —O-tetrafluorophenyl        and —O—C(O)—OR¹⁸;    -   R¹⁷ is selected from —C₁-C₁₀ alkylene-, —C₃-C₈ carbocyclo-,        —O—(C₁-C₈ alkyl)-, -arylene-, —C₁-C₁₀ alkylene-arylene-,        -arylene-C₁-C₁₀ alkylene-, —C₁-C₁₀ alkylene-(C₃-C₈ carbocyclo)-,        —(C₃-C₈ carbocyclo)-C₁-C₁₀ alkylene-, —C₃-C₈ heterocyclo-,        —C₁-C₁₀ alkylene-(C₃-C₈ heterocyclo)-, —(C₃-C₈        heterocyclo)-C₁-C₁₀alkylene-, —(CH₂CH₂O)_(r)—, and        —(CH₂CH₂O)_(r)—CH₂—; r is an integer ranging from 1-10; and    -   R¹⁸ is —C₁-C₈ alkyl or -aryl.

In another aspect, the present invention provides compounds of theformula IIh:

-   -   and pharmaceutically acceptable salts and solvates thereof    -   wherein, independently at each location:    -   R² is selected from —H and —C₁-C₈ alkyl;    -   R³ is selected from —H, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,        —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈        carbocycle), —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈        heterocycle);    -   R⁴ is selected from —H, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,        —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈        carbocycle), —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈        heterocycle) wherein R⁵ is selected from —H and -methyl; or R⁴        and R⁵ join, have the formula —(CR^(a)R^(b))_(n)— wherein R^(a)        and R^(b) are independently selected from —H, —C₁-C₈ alkyl and        —C₃-C₈ carbocycle and n is selected from 2, 3, 4, 5 and 6, and        form a ring with the carbon atom to which they are attached;    -   R⁶ is selected from —H and —C₁-C₈ alkyl;    -   R⁷ is selected from —H, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,        —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈        carbocycle), —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈        heterocycle);    -   each R⁸ is independently selected from —H, —OH, —C₁-C₈ alkyl,        —C₃-C₈ carbocycle and —O—(C₁-C₈ alkyl);    -   R⁹ is selected from —H and —C₁-C₈ alkyl;    -   Z is —O—, —S—, —NH— or —N(R¹⁴)—;    -   R¹¹ is selected from —H, —OH, —NH₂, —NHR¹⁴, —N(R¹⁴)₂, —C₁-C₈        alkyl, —C₃-C₈ carbocycle, —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈        alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈ carbocycle), —C₃-C₈ heterocycle        and —C₁-C₈ alkyl-(C₃-C₈ heterocycle); or R¹¹ is an oxygen atom        which forms a carbonyl unit (C═O) with the carbon atom to which        it is attached and a hydrogen atom on this carbon atom is        replaced by one of the bonds in the (C═O) double bond;    -   each R¹² is independently selected from -aryl and —C₃-C₈        heterocycle;    -   each R¹⁴ is independently —H or —C₁-C₈ alkyl;    -   R¹⁶ is —Yy-Ww-A′        wherein    -   each —W— is independently an Amino Acid unit;    -   —Y— is a Spacer unit;    -   w is an integer ranging from 0 to 12;    -   y is 0, 1 or 2; and    -   -A′ is selected from

wherein

-   -   G is selected from —Cl, —Br, —I, —O-mesyl and —O-tosyl;    -   J is selected from —Cl, —Br, —I, —F, —OH, —O—N-succinimide,        —O-(4-nitrophenyl), —O-pentafluorophenyl, —O-tetrafluorophenyl        and —O—C(O)—OR¹⁸;    -   R¹⁷ is selected from —C₁-C₁₀ alkylene-, —C₃-C₈ carbocyclo-,        —O—(C₁-C₈ alkyl)-, -arylene-, —C₁-C₁₀ alkylene-arylene-,        -arylene-C₁-C₁₀ alkylene-, —C₁-C₁₀ alkylene-(C₃-C₈ carbocyclo)-,        —(C₃-C₈ carbocyclo)-C₁-C₁₀ alkylene-, —C₃-C₈ heterocyclo-,        —C₁-C₁₀ alkylene-(C₃-C₈ heterocyclo)-, —(C₃-C₈        heterocyclo)-C₁-C₁₀ alkylene-, —(CH₂CH₂O)_(r)—, and        —(CH₂CH₂O)_(r)—CH₂—; r is an integer ranging from 1-10; and    -   R¹⁸ is —C₁-C₈ alkyl or -aryl.

In another aspect, the present invention provides compounds of theformula IIi:

-   -   and pharmaceutically acceptable salts and solvates thereof    -   wherein, independently at each location:    -   R² is selected from —H and —C₁-C₈ alkyl;    -   R³ is selected from —H, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,        —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈        carbocycle), —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈        heterocycle);    -   R⁴ is selected from —H, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,        —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈        carbocycle), —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈        heterocycle) wherein R⁵ is selected from —H and -methyl; or R⁴        and R⁵ join, have the formula —(CR^(a)R^(b))_(n)— wherein R^(a)        and R^(b) are independently selected from —H, —C₁-C₈ alkyl and        —C₃-C₈ carbocycle and n is selected from 2, 3, 4, 5 and 6, and        form a ring with the carbon atom to which they are attached;    -   R⁶ is selected from —H and —C₁-C₈ alkyl;    -   R⁷ is selected from —H, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,        —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈        carbocycle), —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈        heterocycle);    -   each R⁸ is independently selected from —H, —OH, —C₁-C₈ alkyl,        —C₃-C₈ carbocycle and —O—(C₁-C₈ alkyl);    -   R⁹ is selected from —H and —C₁-C₈ alkyl;    -   R¹¹ is selected from —H, —OH, —NH₂, —NHR¹⁴, —N(R¹⁴)₂, —C₁-C₈        alkyl, —C₃-C₈ carbocycle, —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈        alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈ carbocycle), —C₃-C₈ heterocycle        and —C₁-C₈ alkyl-(C₃-C₈ heterocycle); or R₁₁ is an oxygen atom        which forms a carbonyl unit (C═O) with the carbon atom to which        it is attached and a hydrogen atom on this carbon atom is        replaced by one of the bonds in the (C═O) double bond;    -   each R¹² is independently selected from -aryl and —C₃-C₈        heterocycle;    -   R¹³ is selected from —H, —OH, —NH₂, —NHR¹⁴, —N(R¹⁴)₂, —C₁-C₈        alkyl, —C₃-C₈ carbocycle, —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈        alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈ carbocycle), —C₃-C₈ heterocycle        and —C₁-C₈ alkyl-(C₃-C₈ heterocycle);    -   each R¹⁴ is independently —H or —C₁-C₈ alkyl;    -   R¹⁶ is —Yy-Ww-A′        wherein    -   each —W— is independently an Amino Acid unit;    -   —Y— is a Spacer unit;    -   w is an integer ranging from 0 to 12;    -   y is 0, 1 or 2; and    -   -A′ is selected from

wherein

-   -   G is selected from —Cl, —Br, —I, —O-mesyl and —O-tosyl;    -   J is selected from —Cl, —Br, —I, —F, —OH, —O—N-succinimide,        —O-(4-nitrophenyl), —O-pentafluorophenyl, —O-tetrafluorophenyl        and —O—C(O)—OR¹⁸;    -   R¹⁷ is selected from —C₁-C₁₀ alkylene-, —C₃-C₈ carbocyclo-,        —O—(C₁-C₈ alkyl)-, -arylene-, —C₁-C₁₀ alkylene-arylene-,        -arylene-C₁-C₁₀ alkylene-, —C₁-C₁₀ alkylene-(C₃-C₈ carbocyclo)-,        —(C₃-C₈ carbocyclo)-C₁-C₁₀ alkylene-, —C₃-C₈ heterocyclo-,        —C₁-C₁₀ alkylene-(C₃-C₈ heterocyclo)-, —(C₃-C₈        heterocyclo)-C₁-C₁₀alkylene-, —(CH₂CH₂O)_(r)—, and        —(CH₂CH₂O)_(r)—CH₂—; r is an integer ranging from 1-10; and    -   R¹⁸ is —C₁-C₈ alkyl or -aryl.

A compound of formula IIa-i or a pharmaceutically acceptable salt orsolvate thereof (a “Drug-Linker Compound”) is useful for treatingcancer, an autoimmune disease or an infectious disease in an animal oruseful as an intermediate for the synthesis of a Drug-Linker-LigandConjugate.

In another aspect, the present invention provides compositionscomprising an effective amount of a Drug-Linker-Ligand Conjugate and apharmaceutically acceptable carrier or vehicle.

In still another aspect, the present invention provides compositionscomprising an effective amount of a Drug-Linker Compound and apharmaceutically acceptable carrier or vehicle.

In yet another aspect, the present invention provides methods forkilling or inhibiting the multiplication of a tumor cell or cancer cell,comprising administering to an animal in need thereof an effectiveamount of a Drug-Linker Compound.

In another aspect, the present invention provides methods for killing orinhibiting the multiplication of a tumor cell or cancer cell, comprisingadministering to an animal in need thereof an effective amount of aDrug-Linker-Ligand Conjugate.

In still another aspect, the invention provides methods for treatingcancer, comprising administering to an animal in need thereof aneffective amount of a Drug-Linker Compound.

In yet another aspect, the invention provides methods for treatingcancer, comprising administering to an animal in need thereof aneffective amount of a Drug-Linker-Ligand Conjugate.

In still another aspect, the invention provides methods for killing orinhibiting the replication of a cell that expresses an auto-immuneantibody, comprising administering to an animal in need thereof aneffective amount of a Drug-Linker Compound.

In another aspect, the invention provides methods for killing orinhibiting the replication of a cell that expresses an auto-immuneantibody, comprising administering to an animal in need thereof aneffective amount of a Drug-Linker-Ligand Conjugate.

In yet another aspect, the invention provides methods for treating anautoimmune disease, comprising administering to an animal in needthereof an effective amount of a Drug-Linker Compound.

In yet another aspect, the invention provides methods for treating anautoimmune disease, comprising administering to an animal in needthereof an effective amount of a Drug-Linker-Ligand Conjugate.

In still another aspect, the invention provides methods for treating aninfectious disease, comprising administering to an animal in needthereof an effective amount of a Drug-Linker Compound.

In still another aspect, the invention provides methods for treating aninfectious disease, comprising administering to an animal in needthereof an effective amount of a Drug-Linker-Ligand Conjugate.

In yet another aspect, the present invention provides methods forpreventing the multiplication of a tumor cell or cancer cell, comprisingadministering to an animal in need thereof an effective amount of aDrug-Linker Compound.

In another aspect, the present invention provides methods for preventingthe multiplication of a tumor cell or cancer cell, comprisingadministering to an animal in need thereof an effective amount of aDrug-Linker-Ligand Conjugate.

In still another aspect, the invention provides methods for preventingcancer, comprising administering to an animal in need thereof aneffective amount of a Drug-Linker Compound.

In yet another aspect, the invention provides methods for preventingcancer, comprising administering to an animal in need thereof aneffective amount of a Drug-Linker-Ligand Conjugate.

In still another aspect, the invention provides methods for preventingthe multiplication of a cell that expresses an auto-immune antibody,comprising administering to an animal in need thereof an effectiveamount of a Drug-Linker Compound.

In another aspect, the invention provides methods for preventing themultiplication of a cell that expresses an auto-immune antibody,comprising administering to an animal in need thereof an effectiveamount of a Drug-Linker-Ligand Conjugate.

In yet another aspect, the invention provides methods for preventing anautoimmune disease, comprising administering to an animal in needthereof an effective amount of a Drug-Linker Compound.

In yet another aspect, the invention provides methods for preventing anautoimmune disease, comprising administering to an animal in needthereof an effective amount of a Drug-Linker-Ligand Conjugate.

In still another aspect, the invention provides methods for preventingan infectious disease, comprising administering to an animal in needthereof an effective amount of a Drug-Linker Compound.

In still another aspect, the invention provides methods for preventingan infectious disease, comprising administering to an animal in needthereof an effective amount of a Drug-Linker-Ligand Conjugate.

In another aspect, the invention provides a Drug-Linker Compound whichcan be used as an intermediate for the synthesis of a Drug-Linker-LigandConjugate.

The present invention may be understood more fully by reference to thefollowing detailed description, Figures and illustrative examples, whichare intended to exemplify non-limiting embodiments of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the cytotoxicity of Compound 49 and Compound 53 against theH3396 cell line. Line -Δ- represents Compound 49 and line -∘- representsCompound 53.

FIG. 2 shows the cytotoxicity of Compounds 64, 65, 68 and 69 against theH3396 cell line. Line -♦- represents Compound 64, line -▪- representsCompound 65, line -▴- represents Compound 68, and line -×- representsCompound 69.

FIG. 3 shows the cytotoxicity of Compounds 64, 65, 68 and 69 against theHCT-116 cell line. Line -♦- represents Compound 64, line -▪- representsCompound 65, line -▴- represents Compound 68, and line -×- representsCompound 69.

FIG. 4 shows the cytotoxicity of Compounds 66 and 68 against the H3396cell line. Line -□- represents Compound 66 and line -*- representsCompound 68.

FIG. 5 shows the cytotoxicity of Compounds 66, 68 and 69 against theKarpas human colorectal cell line. Line -♦- represents Compound 66, line-▴- represents Compound 68, and line -×- represents Compound 69.

FIG. 6 shows the cytotoxicity of Compounds 66 and 67 against the H3396cell line as a function of exposure length. The cells were eitherexposed to the conjugates for the entire duration of the assay withoutwashing (96 hours), or were exposed to the conjugates for 2 hours,washed, and then incubated for an additional 94 hours. At the end of the96 hour period, the cells were pulsed with Alamar Blue to determine cellviability. Line - - represents Compound 66 at 2 h exposure, line -∘-represents Compound 67 at 2 h exposure, line -•- represents Compound 66at 96 h exposure, and line -⋄- represents Compound 67 at 96 h exposure.

FIG. 7 shows the effect of Compounds 66-69 on the growth of L2987 humanlung adenocarcinoma xenograft tumors which were implanted in nude mice.Line -×- represents untreated tumor, line -▾- represents Compound 66,line -♦- represents Compound 68, line -∇- Compound 67, and line -⋄-represents Compound 69.

FIG. 8 shows the effects of Compounds 66-69 on the growth of Karpashuman anaplastic large cell lymphoma xenograft tumors which wereimplanted in nude mice. Line -×- represents untreated tumor, line -Δ-represents Compound 67, line -•- represents Compound 69, line -Δ-represents Compound 66, and line -∘- represents Compound 68.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Examples of an “animal” include, but are not limited to, a human, rat,mouse, guinea pig, monkey, pig, goat, cow, horse, dog, cat, bird andfowl.

“Aryl” refers to a carbocyclic aromatic group Examples of aryl groupsinclude, but are not limited to, phenyl, naphthyl and anthracenyl. Acarbocyclic aromatic group or a heterocyclic aromatic group can beunsubstituted or substituted with one or more groups including, but notlimited to, —C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -aryl, —C(O)R′, —OC(O)R′,—C(O)OR′, —C(O)NH₂, —C(O)NHR′, —C(O)N(R′)₂—NHC(O)R′, —S(O)₂R′, —S(O)R′,—OH, —halogen, —N₃ , —NH₂, —NH(R′), —N(R′)₂ and —CN; where each R′ isindependently selected from —C₁-C₈ alkyl and aryl.

The term “C₁-C₈ alkyl,” as used herein refers to a straight chain orbranched, saturated or unsaturated hydrocarbon having from 1 to 8 carbonatoms. Representative “C₁-C₈ alkyl” groups include, but are not limitedto, -methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, -n-hexyl,-n-heptyl, -n-octyl, -n-nonyl and -n-decyl; while branched C₁-C₈ alkylsinclude, but are not limited to, -isopropyl, -sec-butyl, -isobutyl,-tert-butyl, -isopentyl, 2-methylbutyl, unsaturated C₁-C₈ alkylsinclude, but are not limited to, -vinyl, -allyl, -1-butenyl, -2-butenyl,-isobutylenyl, -1-pentenyl, -2-pentenyl, -3-methyl-1-butenyl,-2-methyl-2-butenyl, -2,3-dimethyl-2-butenyl, 1-hexyl, 2-hexyl,3-hexyl,-acetylenyl, -propynyl, -1-butynyl, -2-butynyl, -1-pentynyl,-2-pentynyl, -3-methyl-1 butynyl.methyl, ethyl, propyl, isopropyl,n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl,neopentyl, n-hexyl, isohexyl, 2-methylpentyl, 3-methylpentyl,2,2-dimethylbutyl, 2,3-dimethylbutyl, 2,2-dimethylpentyl,2,3-dimethylpentyl, 3,3-dimethylpentyl, 2,3,4-trimethylpentyl,3-methylhexyl, 2,2-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl,3,5-dimethylhexyl, 2,4-dimethylpentyl, 2-methylheptyl, 3-methylheptyl,n-heptyl, isoheptyl, n-octyl, and isooctyl. A C₁-C₈ alkyl group can beunsubstituted or substituted with one or more groups including, but notlimited to, —C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -aryl, —C(O)R′, —OC(O)R′,—C(O)OR′, —C(O)NH₂, —C(O)NHR′, —C(O)N(R′)₂—NHC(O)R′, —S(O)₂R′, —S(O)R′,—OH, -halogen, —N₃ , —NH₂, —NH(R′), —N(R′)₂ and —CN; where each R′ isindependently selected from —C₁-C₈ alkyl and aryl.

A “C₃-C₈ carbocycle” is a 3-, 4-, 5-, 6-, 7- or 8-membered saturated orunsaturated non-aromatic carbocyclic ring. Representative C₃-C₈carbocycles include, but are not limited to, -cyclopropyl, -cyclobutyl,-cyclopentyl, -cyclopentadienyl, -cyclohexyl, -cyclohexenyl,-1,3-cyclohexadienyl, -1,4-cyclohexadienyl, -cycloheptyl,-1,3-cycloheptadienyl, -1,3,5-cycloheptatrienyl, -cyclooctyl, and-cyclooctadienyl. A C₃-C₈ carbocycle group can be unsubstituted orsubstituted with one or more groups including, but not limited to,—C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′,—C(O)NH₂, —C(O)NHR′, —C(O)N(R′)₂—NHC(O)R′, —S(O)₂R′, —S(O)R′, —OH,-halogen, —N₃, —NH₂, —NH(R′), —N(R′)₂ and —CN; where each R′ isindependently selected from —C₁-C₈ alkyl and aryl.

A “C₃-C₈ carbocyclo” refers to a C₃-C₈ carbocycle group defined abovewherein one of the carbocycle groups hydrogen atoms is replaced with abond.

A “C₁-C₁₀ alkylene” is a straight chain, saturated hydrocarbon group ofthe formula —(CH₂)₁₋₁₀—. Examples of a C₁-C₁₀ alkylene includemethylene, ethylene, propylene, butylene, pentylene, hexylene,heptylene, ocytylene, nonylene and decalene.

An “arylene” is an aryl group which has two covalent bonds and can be inthe ortho, meta, or para configurations as shown in the followingstructures:

in which the phenyl group can be unsubstituted or substituted with up tofour groups including, but not limited to, —C₁-C₈ alkyl, —O—(C₁-C₈alkyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂, —C(O)NHR′,—C(O)N(R′)₂—NHC(O)R′, —S(O)₂R′, —S(O)R′, —OH, —halogen, —N₃ , —NH₂,—NH(R′), —N(R′)₂ and —CN; where each R′ is independently selected from—C₁-C₈ alkyl and aryl.

A “C₃-C₈ heterocycle” refers to an aromatic or non-aromatic C₃-C₈carbocycle in which one to four of the ring carbon atoms areindependently replaced with a heteroatom from the group consisting of O,S and N. Representative examples of a C₃-C₈ heterocycle include, but arenot limited to, benzofuranyl, benzothiophene, indolyl, benzopyrazolyl,coumarinyl, isoquinolinyl, pyrrolyl, thiophenyl, furanyl, thiazolyl,imidazolyl, pyrazolyl, triazolyl, quinolinyl, pyrimidinyl, pyridinyl,pyridonyl, pyrazinyl, pyridazinyl, isothiazolyl, isoxazolyl andtetrazolyl. A C₃-C₈ Heterocycle can be unsubstituted or substituted withup to seven groups including, but not limited to, —C₁-C₈ alkyl,—O—(C₁-C₈ alkyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂,—C(O)NHR′, —C(O)N(R′)₂—NHC(O)R′, —S(O)₂R′, —S(O)R′, —OH, -halogen, —N₃ ,—NH₂, —NH(R′), —N(R′)₂ and —CN; where each R′ is independently selectedfrom —C₁-C₈ alkyl and aryl.

“C₃-C₈ heterocyclo” refers to a C₃-C₈ heterocycle group defined abovewherein one of the heterocycle groups hydrogen atoms is replaced with abond. A C₃-C₈ heterocyclo can be unsubstituted or substituted with up tosix groups including, but not limited to, —C₁-C₈ alkyl, —O—(C₁-C₈alkyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂, —C(O)NHR′,—C(O)N(R′)₂—NHC(O)R′, —S(O)₂R′, —S(O)R′, —OH, -halogen, —N₃ , —NH₂,—NH(R′), —N(R′)₂ and —CN; where each R′ is independently selected from—C₁-C₈ alkyl and aryl.

A “Compound of the Invention” is a Drug-Linker Compound or aDrug-Linker-Ligand Conjugate.

In one embodiment, the Compounds of the Invention are in isolated orpurified form. As used herein, “isolated” means separated from othercomponents of (a) a natural source, such as a plant or animal cell orcell culture, or (b) a synthetic organic chemical reaction mixture. Asused herein, “purified” means that when isolated, the isolate containsat least 95%, preferably at least 98%, of a Compound of the Invention byweight of the isolate.

Examples of a “Hydroxyl protecting group” include, but are not limitedto, methoxymethyl ether, 2-methoxyethoxymethyl ether, tetrahydropyranylether, benzyl ether, p-methoxybenzyl ether, trimethylsilyl ether,triisopropyl silyl ether, t-butyldimethyl silyl ether, triphenylmethylsilyl ether, acetate ester, substituted acetate esters, pivaloate,benzoate, methanesulfonate and p-toluenesulfonate.

“Leaving group” refers to a functional group that can be substituted byanother functional group. Such leaving groups are well known in the art,and examples include, but are not limited to, a halide (e.g., chloride,bromide, iodide), methanesulfonyl (mesyl), p-toluenesulfonyl (tosyl),trifluoromethylsulfonyl (triflate), and trifluoromethylsulfonate.

The term “antibody,” as used herein, refers to a full-lengthimmunoglobulin molecule or an immunologically active portion of afull-length immunoglobulin molecule, i.e., a molecule that contains anantigen binding site that immunospecifically binds an antigen of atarget of interest or part thereof, such targets including but notlimited to, cancer cell or cells that produce auto-immune antibodiesassociated with an autoimmune disease. The immunoglobulin disclosedherein can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class(e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass ofimmunoglobulin molecule. The immunoglobulins can be derived from anyspecies. Preferably, however, the immunoglobulin is of human, murine, orrabbit origin. Antibodies useful in the invention are preferablymonoclonal, and include, but are not limited to, polyclonal, monoclonal,bispecific, human, humanized or chimeric antibodies, single chainantibodies, Fv, Fab fragments, F(ab′) fragments, F(ab′)₂ fragments,fragments produced by a Fab expression library, anti-idiotypic (anti-Id)antibodies, CDR's, and epitope-binding fragments of any of the abovewhich immunospecifically bind to cancer cell antigens, viral antigens ormicrobial antigens.

The phrase “pharmaceutically acceptable salt,” as used herein, refers topharmaceutically acceptable organic or inorganic salts of a Compound ofthe Invention. The Compounds of the Invention contain at least one aminogroup, and accordingly acid addition salts can be formed with this aminogroup. Preferred salts include, but are not limited, to sulfate,citrate, acetate, oxalate, chloride, bromide, iodide, nitrate,bisulfate, phosphate, acid phosphate, isonicotinate, lactate,salicylate, acid citrate, tartrate, oleate, tannate, pantothenate,bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate,gluconate, glucaronate, saccharate, formate, benzoate, glutamate,methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate,and pamoate (i.e., 1,1′-methylene-bis-(2-hydroxy-3-naphthoate)) salts. Apharmaceutically acceptable salt may involve the inclusion of anothermolecule such as an acetate ion, a succinate ion or other counterion.The counterion may be any organic or inorganic moiety that stabilizesthe charge on the parent compound. Furthermore, a pharmaceuticallyacceptable salt may have more than one charged atom in its structure.Instances where multiple charged atoms are part of the pharmaceuticallyacceptable salt can have multiple counterions. Hence, a pharmaceuticallyacceptable salt can have one or more charged atoms and/or one or morecounterion.

“Pharmaceutically acceptable solvate” refers to an association of one ormore solvent molecules and a Compound of the Invention. Examples ofsolvents that form pharmaceutically acceptable solvates include, but arenot limited to, water, isopropanol, ethanol, methanol, DMSO, ethylacetate, acetic acid, and ethanolamine.

In the context of cancer, the term “treating” includes any or all of:preventing growth of tumor cells or cancer cells, preventing replicationof tumor cells or cancer cells, lessening of overall tumor burden andameliorating one or more symptoms associated with the disease.

In the context of an autoimmune disease, the term “treating” includesany or all of: preventing replication of cells associated with anautoimmune disease state including, but not limited to, cells capable ofproducing an autoimmune antibody, lessening the autoimmune-antibodyburden and ameliorating one or more symptoms of an autoimmune disease.

In the context of an infectious disease, the term “treating” includesany or all of: preventing the growth, multiplication or replication ofthe pathogen that causes the infectious disease and ameliorating one ormore symptoms of an infectious disease.

The following abbreviations are used herein and have the indicateddefinitions: AE is auristatin E, Boc is N-(t-butoxycarbonyl), cit iscitrulline, dap is dolaproine, DCC is 1,3-dicyclohexylcarbodiimide, DCMis dichloromethane, DEA is diethylamine, DEAD isdiethylazodicarboxylate, DEPC is diethylphosphorylcyanidate, DIAD isdiisopropylazodicarboxylate, DIEA is N,N-diisopropylethylamine, dil isdolaisoleuine, DMAP is 4-dimethylaminopyridine, DME is ethyleneglycoldimethyl ether (or 1,2-dimethoxyethane), DMF is N,N-dimethylformamide,DMSO is dimethylsulfoxide, doe is dolaphenine, dov isN,N-dimethylvaline, DTNB is 5,5′-dithiobis(2-nitrobenzoic acid), DTPA isdiethylenetriaminepentaacetic acid, DTT is dithiothreitol, EDCI is1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, EEDQ is2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline, ES-MS is electrospraymass spectrometry, EtOAc is ethyl acetate, Fmoc isN-(9-fluorenylmethoxycarbonyl), gly is glycine, HATU isO-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate, HOBt is 1-hydroxybenzotriazole, HPLC is highpressure liquid chromatography, ile is isoleucine, lys is lysine, MeCNis acetonitrile, MeOH is methanol, Mtr is 4-anisyldiphenylmethyl (or4-methoxytrityl), nor is (1S,2R)-(+)-norephedrine, PAB is p-aminobenzyl,PBS is phosphate-buffered saline (pH 7.4), PEG is polyethylene glycol,Ph is phenyl, Pnp is p-nitrophenyl, MC is 6-maleimidocaproyl, Ph isphenyl, phe is L-phenylalanine, PyBrop isbromo-tris-pyrrolidino-phosphonium hexafluorophosphate, SEC issize-exclusion chromatography, Su is succinimide, TFA is trifluoroaceticacid, TLC is thin layer chromatography, UV is ultraviolet, val isvaline.

Drug-Linker-Ligand Conjugates

As stated above, the invention provides compounds of the formula Ia:

-   -   and pharmaceutically acceptable salts and solvates thereof        wherein,    -   L- is a Ligand unit;    -   -A- is a Stretcher unit;    -   a is 0 or 1;    -   each —W— is independently an Amino Acid unit;    -   —Y— is a Spacer unit;    -   w is an integer ranging from 0 to 12;    -   y is 0, 1 or 2;    -   p ranges from 1 to about 20; and    -   -D is a Drug unit of the formula

-   -   wherein, independently at each location:    -   R² is selected from —H and —C₁-C₈ alkyl;    -   R³ is selected from —H, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,        —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈        carbocycle), —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈        heterocycle);    -   R⁴ is selected from —H, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,        —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈        carbocycle), —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈        heterocycle) wherein R⁵ is selected from —H and -methyl; or R⁴        and R⁵ join, have the formula —(CR^(a)R^(b))_(n)— wherein R^(a)        and R^(b) are independently selected from —H, —C₁-C₈ alkyl and        —C₃-C₈ carbocycle and n is selected from 2, 3, 4, 5 and 6, and        form a ring with the carbon atom to which they are attached;    -   R⁶ is selected from —H and —C₁-C₈ alkyl;    -   R⁷ is selected from —H, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,        —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈        carbocycle), —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈        heterocycle);    -   each R⁸ is independently selected from —H, —OH, —C₁-C₈ alkyl,        —C₃-C₈ carbocycle and —O—(C₁-C₈ alkyl);    -   R⁹ is selected from —H and —C₁-C₈ alkyl;    -   R¹⁰ is selected from

-   -   Z is —O—, —S—, —NH— or —N(R¹⁴)—;    -   R¹¹ is selected from —H, —OH, —NH₂, —NHR¹⁴, —N(R¹⁴)₂, —C₁-C₈        alkyl, —C₃-C₈ carbocycle, —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈        alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈ carbocycle), —C₃-C₈ heterocycle        and —C₁-C₈ alkyl-(C₃-C₈ heterocycle); or R¹¹ is an oxygen atom        which forms a carbonyl unit (C═O) with the carbon atom to which        it is attached and a hydrogen atom on this carbon atom is        replaced by one of the bonds in the (C═O) double bond;    -   each R¹² is independently selected from -aryl and —C₃-C₈        heterocycle;    -   R¹³ is selected from —H, —OH, —NH₂, —NHR¹⁴, —N(R¹⁴)₂, —C₁-C₈        alkyl, —C₃-C₈ carbocycle, —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈        alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈ carbocycle), C₃-C₈ heterocycle        and —C₁-C₈ alkyl-(C₃-C₈ heterocycle); and    -   each R¹⁴ is independently —H or —C₁-C₈ alkyl.

In one embodiment R¹⁰ is selected from

In another embodiment, w is an integer ranging from 2 to 12.

In another embodiment, p ranges from 1 to about 8.

In another embodiment, p ranges from 1 to about 3.

In another embodiment, p ranges from about 3 to about 5.

In still another embodiment, p ranges from about 7 to about 9.

In another embodiment, p is about 8.

In another embodiment, p is about 4.

In a further embodiment, p is about 2.

Illustrative classes of compounds of formula Ia have the structures:

and pharmaceutically acceptable salts and solvates thereof,where L- is a Ligand unit, E is —CH₂— or —CH₂CH₂O—; e is an integerranging either from 0-10 when E is —CH₂—, or from 1-10 when E is—CH₂CH₂—O—; F is —CH—; f is 0 or 1; and p ranges from 1 to about 20.

In another embodiment, p ranges from 1 to about 8.

In another embodiment, p ranges from 1 to about 3.

In another embodiment, p ranges from about 3 to about 5.

In still another embodiment, p ranges from about 7 to about 9.

In another embodiment, p is about 8.

In another embodiment, p is about 4.

In another embodiment L is cBR96, cAC10 or 1F6.

Illustrative compounds of formula Ia have the structure:

and pharmaceutically acceptable salts and solvates thereof,where p ranges from about 7 to about 9.

In one embodiment p ranges from 1 to about 3.

In another embodiment, p ranges from about 3 to about 5.

In another embodiment, p is about 8.

In yet another embodiment, p is about 4.

In a further embodiment, p is about 2.

In another aspect, the present invention provides compounds of generalformula Ib:

-   -   and pharmaceutically acceptable salts and solvates thereof    -   wherein,    -   L- is a Ligand unit;    -   -A- is a Stretcher unit;    -   a is 0 or 1;    -   each —W— is independently an Amino Acid unit;    -   —Y— is a Spacer unit;    -   w is an integer ranging from 0 to 12;    -   y is 0, 1 or 2;    -   p ranges from 1 to about 20; and    -   -D is a Drug unit of the formula

-   -   wherein, independently at each location:    -   R¹ is selected from —H, —C₁-C₈ alkyl and —C₃-C₈ carbocycle; and        R² is selected from —H and —C₁-C₈ alkyl; or R¹ and R² join, have        the formula —(CR^(a)R^(b))_(n)— wherein R^(a) and R^(b) are        independently selected from —H, —C₁-C₈ alkyl and —C₃-C₈        carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a        ring with the nitrogen atom to which they are attached;    -   R³ is selected from —H, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,        —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈        carbocycle), —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈        heterocycle);    -   R⁴ is selected from —H, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,        —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈        carbocycle), —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈        heterocycle) wherein R⁵ is selected from —H and -methyl; or R⁴        and R⁵ join, have the formula —(CR^(a)R^(b))_(n)— wherein R^(a)        and R^(b) are independently selected from —H, —C₁-C₈ alkyl and        —C₃-C₈ carbocycle and n is selected from 2, 3, 4, 5 and 6, and        form a ring with the carbon atom to which they are attached;    -   R⁶ is selected from —H and —C₁-C₈ alkyl;    -   R⁷ is selected from —H, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,        —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈        carbocycle), —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈        heterocycle);    -   each R⁸ is independently selected from —H, —OH, —C₁-C₈ alkyl,        —C₃-C₈ carbocycle and —O—(C₁-C₈ alkyl);    -   R⁹ is selected from —H and —C₁-C₈ alkyl;    -   R¹⁰ is selected from

-   -   X is —O—, —S—, —NH— or —N(R¹⁴)—, where X is bonded to Y when y        is 1 or 2, or X is bonded to W when y is 0;    -   Z is —O—, —S—, —NH— or —N(R¹⁴)—;    -   R¹¹ is selected from —H, —OH, —NH₂, —NHR¹⁴, —N(R¹⁴)₂, —C₁-C₈        alkyl, —C₃-C₈ carbocycle, —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈        alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈ carbocycle), —C₃-C₈ heterocycle        and —C₁-C₈ alkyl-(C₃-C₈ heterocycle); or R¹¹ is an oxygen atom        which forms a carbonyl unit (C═O) with the carbon atom to which        it is attached and a hydrogen atom on this carbon atom is        replaced by one of the bonds in the (C═O) double bond;    -   each R¹² is independently selected from -aryl and —C₃-C₈        heterocycle;    -   R¹³ is selected from —H, —OH, —NH₂, —NHR¹⁴, —N(R¹⁴)₂, —C₁-C₈        alkyl, —C₃-C₈ carbocycle, —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈        alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈ carbocycle), C₃-C₈ heterocycle        and —C₁-C₈ alkyl-(C₃-C₈ heterocycle);    -   each R¹⁴ is independently —H or —C₁-C₈ alkyl; and    -   R¹⁵ is -arylene-, —C₃-C₈ carbocyclo- or —C₃-C₈ heterocyclo-.

In one embodiment, when R¹ is —H, R¹⁰ is selected from:

In another embodiment, w is an integer ranging from 2 to 12.

In another embodiment, p ranges from 1 to about 8.

In another embodiment, p ranges from 1 to about 3.

In another embodiment, p ranges from about 3 to about 5.

In still another embodiment, p ranges from about 7 to about 9.

In another embodiment, p is about 8.

In another embodiment, p is about 4.

In a further embodiment, p is about 2.

Illustrative classes of compounds of formula Ib have the structure:

pharmaceutically acceptable salts and solvates thereof,

-   -   where L- is Ligand unit, E is —CH₂— or —CH₂CH₂O—; e is an        integer ranging either from 0-10 when E is —CH₂—, or 1-10 when E        is —CH₂CH₂—O—; F is —CH₂—; f is 0 or 1; and p ranges from 1 to        about 20.

In another embodiment, p ranges from 1 to about 8.

In another embodiment, p ranges from 1 to about 3.

In another embodiment, p ranges from about 3 to about 5.

In still another embodiment, p ranges from about 7 to about 9.

In another embodiment, p is about 8.

In another embodiment, p is about 4.

In a further embodiment, p is about 2.

In another embodiment L is cBR96, cAC10 or 1F6.

Illustrative compounds of formula Ib have the structure:

and pharmaceutically acceptable salts and solvates thereof,where p ranges from about 7 to about 9.

In one embodiment p ranges from 1 to about 3.

In another embodiment, p ranges from about 3 to about 5.

In another embodiment, p is about 8.

In yet another embodiment, p is about 4.

In a further embodiment, p is about 2.

In another aspect, the present invention provides compounds of generalformula Ic:

-   -   L- is a Ligand unit;    -   -A- is a Stretcher unit;    -   a is 0 or 1;    -   each —W— is independently an Amino Acid unit;    -   w is an integer ranging from 0 to 12;    -   each n is independently 0 or 1;    -   p ranges from 1 to about 20; and    -   each -D is independently:    -   (a) a Drug unit of the formula:

-   -   wherein, independently at each location:    -   R¹ is selected from —H, —C₁-C₈ alkyl and —C₃-C₈ carbocycle; and        R² is selected from —H and —C₁-C₈ alkyl; or R¹ and R² join, have        the formula —(CR^(a)R^(b))_(n)— wherein R^(a) and R^(b) are        independently selected from —H, —C₁-C₈ alkyl and —C₃-C₈        carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a        ring with the nitrogen atom to which they are attached;    -   R³ is selected from —H, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,        —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈        carbocycle), —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈        heterocycle);    -   R⁴ is selected from —H, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,        —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈        carbocycle), —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈        heterocycle) wherein R⁵ is selected from —H and -methyl; or R⁴        and R⁵ join, have the formula —(CR^(a)R^(b))_(n)— wherein R^(a)        and R^(b) are independently selected from —H, —C₁-C₈ alkyl and        —C₃-C₈ carbocycle and n is selected from 2, 3, 4, 5 and 6, and        form a ring with the carbon atom to which they are attached;    -   R⁶ is selected from —H and —C₁-C₈ alkyl;    -   R⁷ is selected from —H, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,        —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈        carbocycle), —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈        heterocycle);    -   each R⁸ is independently selected from —H, —OH, —C₁-C₈ alkyl,        —C₃-C₈ carbocycle and —O—(C₁-C₈ alkyl);    -   R⁹ is selected from —H and —C₁-C₈ alkyl;    -   R¹⁰ is selected from

-   -   X is —O—, —S—, —NH— or —N(R¹⁴)—, where X is bonded to —C(O)—        when y is 1 or 2, or X is bonded to —CH₂— when n is 0;    -   Z is —O—, —S—, —NH— or —N(R¹⁴)—;    -   R¹¹ is selected from —H, —OH, —NH₂, —NHR¹⁴, —N(R¹⁴)₂, —C₁-C₈        alkyl, —C₃-C₈ carbocycle, —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈        alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈ carbocycle), —C₃-C₈ heterocycle        and —C₁-C₈ alkyl-(C₃-C₈ heterocycle); or R¹¹ is an oxygen atom        which forms a carbonyl unit (C═O) with the carbon atom to which        it is attached and a hydrogen atom on this carbon atom is        replaced by one of the bonds in the (C═O) double bond;    -   each R¹² is independently selected from -aryl and —C₃-C₈        heterocycle;    -   R¹³ is selected from —H, —OH, —NH₂, —NHR¹⁴, —N(R¹⁴)₂, —C₁-C₈        alkyl, —C₃-C₈ carbocycle, —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈        alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈ carbocycle), C₃-C₈ heterocycle        and —C₁-C₈ alkyl-(C₃-C₈ heterocycle);    -   each R¹⁴ is independently —H or —C₁-C₈ alkyl; and    -   R¹⁵ is -arylene-, —C₃-C₈ carbocyclo- or —C₃-C₈ heterocyclo-; or    -   (b) a Drug unit of the formula:

-   -   wherein, independently at each location:    -   R² is selected from —H and —C₁-C₈ alkyl;    -   R³ is selected from —H, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,        —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈        carbocycle), —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈        heterocycle);    -   R⁴ is selected from —H, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,        —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈        carbocycle), —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈        heterocycle) wherein R⁵ is selected from —H and -methyl; or R⁴        and R⁵ join, have the formula —(CR^(a)R^(b))_(n)— wherein R^(a)        and R^(b) are independently selected from —H, —C₁-C₈ alkyl and        —C₃-C₈ carbocycle and n is selected from 2, 3, 4, 5 and 6, and        form a ring with the carbon atom to which they are attached;    -   R⁶ is selected from —H and —C₁-C₈ alkyl;    -   R⁷ is selected from —H, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,        —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈        carbocycle), —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈        heterocycle);    -   each R⁸ is independently selected from —H, —OH, —C₁-C₈ alkyl,        —C₃-C₈ carbocycle and —O—(C₁-C₈ alkyl);    -   R⁹ is selected from —H and —C₁-C₈ alkyl;    -   R¹⁰ is selected from

-   -   Z is —O—, —S—, —NH— or —N(R¹⁴)—;    -   R¹¹ is selected from —H, —OH, —NH₂, —NHR¹⁴, —N(R¹⁴)₂, —C₁-C₈        alkyl, —C₃-C₈ carbocycle, —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈        alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈ carbocycle), —C₃-C₈ heterocycle        and —C₁-C₈ alkyl-(C₃-C₈ heterocycle); or R¹¹ is an oxygen atom        which forms a carbonyl unit (C═O) with the carbon atom to which        it is attached and a hydrogen atom on this carbon atom is        replaced by one of the bonds in the (C═O) double bond;    -   each R¹² is independently selected from -aryl and —C₃-C₈        heterocycle;    -   R¹³ is selected from —H, —OH, —NH₂, —NHR¹⁴, —N(R¹⁴)₂, —C₁-C₈        alkyl, —C₃-C₈ carbocycle, —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈        alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈ carbocycle), C₃-C₈ heterocycle        and —C₁-C₈ alkyl-(C₃-C₈ heterocycle); and    -   each R¹⁴ is independently —H or —C₁-C₈ alkyl.

In one embodiment, when the drug unit has the formula:

and R¹ is —H, R¹⁰ is selected from

In another embodiment, when the drug unit has the formula:

-   R¹⁰ is selected from

In another embodiment, w is an integer ranging from 2 to 12.

In another embodiment, p ranges from 1 to about 8.

In another embodiment, p ranges from 1 to about 3.

In another embodiment, p ranges from about 3 to about 5.

In still another embodiment, p ranges from about 7 to about 9.

In another embodiment, p is about 8.

In another embodiment, p is about 4.

In a further embodiment, p is about 2.

An illustrative compound of formula Ic has the structure:

wherein where L- is Ligand unit, E is —CH₂— or —CH₂CH₂O—; e is aninteger ranging either from 0-10 when E is —CH₂—, or 1-10 when E is—CH₂CH₂—O—; F is —CH₂—; f is 0 or 1; and p ranges from 1 to about 20.

In another embodiment, p ranges from 1 to about 8.

In another embodiment, p ranges from 1 to about 3.

In another embodiment, p ranges from about 3 to about 5.

In still another embodiment, p ranges from about 7 to about 9.

In another embodiment, p is about 8.

In another embodiment, p is about 4.

In a further embodiment, p is about 2.

In another embodiment L is cBR96, cAC10 or 1F6.

The Drug-Linker-Ligand Conjugates are useful for treating or preventingcancer, an autoimmune disease or an infectious disease in an animal.

It is understood that p is the average number of -A_(a)-W_(w)—Y_(y)-Dunits per ligand in a Drug-Linker-Ligand Conjugate of formulas Ia, Iband Ic.

In one embodiment p ranges from 1 to 15.

In another embodiment p ranges from 1 to 10.

In another embodiment, p ranges from 1 to about 8.

In a further embodiment p ranges from 1 to about 5.

In another embodiment p ranges from 1 to about 3.

In one embodiment p ranges from about 3 to about 5.

In one embodiment p ranges from about 7 to about 9.

In another embodiment p is about 8.

In yet another embodiment p is about 4.

In still another embodiment p is about 2.

The Drug-Linker-Ligand Conjugates of formulas Ia, Ib and Ic may exist asmixtures, wherein each component of a mixture has a different p value.For example, a Drug-Linker-Ligand Conjugate may exist as a mixture oftwo separate Conjugates, one Conjugate component wherein p is 7 and theother Conjugate component wherein p is 8.

In one embodiment, a Drug-Linker-Ligand Conjugate exists as a mixture ofthree separate conjugates wherein p for the three separate conjugates is1, 2, and 3, respectively.

In another embodiment, a Drug-Linker-Ligand Conjugate exists as amixture of three separate conjugates wherein p for the three separateconjugates is 3, 4, and 5, respectively.

In another embodiment, a Drug-Linker-Ligand Conjugate exists as amixture of three separate conjugates wherein p for the three separateconjugates is 5, 6, and 7, respectively.

In still another embodiment, a Drug-Linker-Ligand Conjugate exists as amixture of three separate conjugates wherein p for the three separateconjugates is 7, 8, and 9, respectively.

In yet another embodiment, a Drug-Linker-Ligand Conjugate exists as amixture of three separate conjugates wherein p for the three separateconjugates is 9, 10, and 11, respectively.

In still another embodiment, a Drug-Linker-Ligand Conjugate exists as amixture of three separate conjugates wherein p for the three separateconjugates is 11, 12, and 13, respectively.

In another embodiment, a Drug-Linker-Ligand Conjugate exists as amixture of three separate conjugates wherein p for the three separateconjugates is 13, 14, and 15, respectively.

Drug-Linker Compounds

The present invention provides compounds of the formula IIa:

-   -   and pharmaceutically acceptable salts and solvates thereof    -   wherein, independently at each location:    -   R¹ is selected from —H, —C₁-C₈ alkyl and —C₃-C₈ carbocycle; and        R² is selected from —H and —C₁-C₈ alkyl; or R¹ and R² join, have        the formula —(CR^(a)R^(b))_(n)— wherein R^(a) and R^(b) are        independently selected from —H, —C₁-C₈ alkyl and —C₃-C₈        carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a        ring with the nitrogen atom to which they are attached;    -   R³ is selected from —H, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,        —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈        carbocycle), —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈        heterocycle);    -   R⁴ is selected from —H, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,        —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈        carbocycle), —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈        heterocycle) wherein R⁵ is selected from —H and -methyl; or R⁴        and R⁵ join, have the formula —(CR^(a)R^(b))_(n)— wherein R^(a)        and R^(b) are independently selected from —H, —C₁-C₈ alkyl and        —C₃-C₈ carbocycle and n is selected from 2, 3, 4, 5 and 6, and        form a ring with the carbon atom to which they are attached;    -   R⁶ is selected from —H and —C₁-C₈ alkyl;    -   R⁷ is selected from —H, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,        —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈        carbocycle), —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈        heterocycle);    -   each R⁸ is independently selected from —H, —OH, —C₁-C₈ alkyl,        —C₃-C₈ carbocycle and —O—(C₁-C₈ alkyl);    -   R⁹ is selected from —H and —C₁-C₈ alkyl; X is —O—, —S—, —NH— or        —N(R¹⁴)—, where X is bonded to Y when y is 1 or 2, or X is        bonded to W when y is 0;    -   R¹¹ is selected from —H, —OH, —NH₂, —NHR¹⁴, —N(R¹⁴)₂, —C₁-C₈        alkyl, —C₃-C₈ carbocycle, —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈        alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈ carbocycle), —C₃-C₈ heterocycle        and —C₁-C₈ alkyl-(C₃-C₈ heterocycle); or R¹¹ is an oxygen atom        which forms a carbonyl unit (C═O) with the carbon atom to which        it is attached and a hydrogen atom on this carbon atom is        replaced by one of the bonds in the (C═O) double bond;    -   each R¹² is independently selected from -aryl and —C₃-C₈        heterocycle;    -   each R¹⁴ is independently —H or —C₁-C₈ alkyl;    -   R¹⁶ is —Yy-Ww-A′        wherein    -   each —W— is independently an Amino Acid unit;    -   —Y— is a Spacer unit;    -   w is an integer ranging from 0 to 12;    -   y is 0, 1 or 2; and    -   -A′ is selected from

wherein

-   -   G is selected from —Cl, —Br, —I, —O-mesyl and —O-tosyl;    -   J is selected from —Cl, —Br, —I, —F, —OH, —O—N-succinimide,        -O-(4-nitrophenyl), —O-pentafluorophenyl, —O-tetrafluorophenyl        and —O—C(O)—OR¹⁸;    -   R¹⁷ is selected from —C₁-C₁₀ alkylene-, —C₃-C₈ carbocyclo-,        —O—(C₁-C₈ alkyl)-, -arylene-, —C₁-C₁₀ alkylene-arylene-,        -arylene-C₁-C₁₀ alkylene-, —C₁-C₁₀ alkylene-(C₃-C₈ carbocyclo)-,        —(C₃-C₈ carbocyclo)-C₁-C₁₀ alkylene-, —C₃-C₈ heterocyclo-,        —C₁-C₁₀ alkylene-(C₃-C₈ heterocyclo)-, —(C₃-C₈        heterocyclo)-C₁-C₁₀ alkylene-, —(CH₂CH₂O)_(r)—, and        —(CH₂CH₂O)_(r)—CH₂—; r is an integer ranging from 1-10; and    -   R¹⁸ is —C₁-C₈ alkyl or -aryl.

An illustrative compound of formula IIa has the structure:

-   -   and pharmaceutically acceptable salts and solvates thereof

In another aspect, the present invention provides compounds of theformula IIb:

-   -   and pharmaceutically acceptable salts and solvates thereof    -   wherein, independently at each location:    -   R¹ is selected from —H, —C₁-C₈ alkyl and —C₃-C₈ carbocycle; and        R² is selected from —H and —C₁-C₈ alkyl; or R¹ and R² join, have        the formula —(CR^(a)R^(b))_(n)— wherein R^(a) and R^(b) are        independently selected from —H, —C₁-C₈ alkyl and —C₃-C₈        carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a        ring with the nitrogen atom to which they are attached;    -   R³ is selected from —H, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,        —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈        carbocycle), —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈        heterocycle);    -   R⁴ is selected from —H, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,        —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈        carbocycle), —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈        heterocycle) wherein R⁵ is selected from —H and -methyl; or R⁴        and R⁵ join, have the formula —(CR^(a)R^(b))_(n)— wherein R^(a)        and R^(b) are independently selected from —H, —C₁-C₈ alkyl and        —C₃-C₈ carbocycle and n is selected from 2, 3, 4, 5 and 6, and        form a ring with the carbon atom to which they are attached;    -   R⁶ is selected from —H and —C₁-C₈ alkyl;    -   R⁷ is selected from —H, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,        —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈        carbocycle), —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈        heterocycle);    -   each R⁸ is independently selected from —H, —OH, —C₁-C₈ alkyl,        —C₃-C₈ carbocycle and —O—(C₁-C₈ alkyl);    -   R⁹ is selected from —H and —C₁-C₈ alkyl;    -   X is —O—, —S—, —NH— or —N(R¹⁴)—;    -   R¹¹ is selected from —H, —OH, —NH₂, —NHR¹⁴, —N(R¹⁴)₂, —C₁-C₈        alkyl, —C₃-C₈ carbocycle, —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈        alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈ carbocycle), —C₃-C₈ heterocycle        and —C₁-C₈ alkyl-(C₃-C₈ heterocycle); or R¹¹ is an oxygen atom        which forms a carbonyl unit (C═O) with the carbon atom to which        it is attached and a hydrogen atom on this carbon atom is        replaced by one of the bonds in the (C═O) double bond;    -   R¹³ is selected from hydrogen, —OH, —NH₂, —NHR¹⁴, —N(R¹⁴)₂,        C₁-C₈ alkyl, C₃-C₈ carbocycle, —O—(C₁-C₈ alkyl), -aryl,        alkyl-aryl, alkyl-(C₃-C₈ carbocycle), C₃-C₈ heterocycle and        alkyl-(C₃-C₈ heterocycle);    -   each R¹⁴ is independently —H or —C₁-C₈ alkyl;    -   R¹⁵ is -arylene-, —C₃-C₈ carbocyclo- or —C₃-C₈ heterocyclo-;    -   R¹⁶ is —Yy-Ww-A′        wherein    -   each —W— is independently an Amino Acid unit;    -   —Y— is a Spacer unit;    -   w is an integer ranging from 0 to 12;    -   y is 0, 1 or 2; and    -   -A′ is selected from

wherein

-   -   G is selected from —Cl, —Br, —I, —O-mesyl and —O-tosyl;    -   J is selected from —Cl, —Br, —I, —F, —OH, —O—N-succinimide,        —O-(4-nitrophenyl), —O-pentafluorophenyl, —O-tetrafluorophenyl        and —O—C(O)—OR¹⁸;    -   R¹⁷ is selected from —C₁-C₁₀ alkylene-, —C₃-C₈ carbocyclo-,        —O—(C₁-C₈ alkyl)-, -arylene-, —C₁-C₁₀ alkylene-arylene-,        -arylene-C₁-C₁₀ alkylene-, —C₁-C₁₀ alkylene-(C₃-C₈ carbocyclo)-,        —(C₃-C₈ carbocyclo)-C₁-C₁₀ alkylene-, —C₃-C₈ heterocyclo-,        —C₁-C₁₀ alkylene-(C₃-C₈ heterocyclo)-, —(C₃-C₈        heterocyclo)-C₁-C₁₀alkylene-, —(CH₂CH₂O)_(r)—, and        —(CH₂CH₂O)_(r)—CH₂—; r is an integer ranging from 1-10; and    -   R¹⁸ is —C₁-C₈ alkyl or -aryl.

In another aspect, the present invention provides compounds of theformula IIc:

-   -   and pharmaceutically acceptable salts and solvates thereof    -   wherein, independently at each location:    -   R¹ is selected from —H, —C₁-C₈ alkyl and —C₃-C₈ carbocycle; and        R² is selected from —H and —C₁-C₈ alkyl; or R¹ and R² join, have        the formula —(CR^(a)R^(b))_(n)— wherein R^(a) and R^(b) are        independently selected from —H, —C₁-C₈ alkyl and —C₃-C₈        carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a        ring with the nitrogen atom to which they are attached;    -   R³ is selected from —H, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,        —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈        carbocycle), —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈        heterocycle);    -   R⁴ is selected from —H, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,        —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈        carbocycle), —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈        heterocycle) wherein R⁵ is selected from —H and -methyl; or R⁴        and R⁵ join, have the formula —(CR^(a)R^(b))_(n)— wherein R^(a)        and R^(b) are independently selected from —H, —C₁-C₈ alkyl and        —C₃-C₈ carbocycle and n is selected from 2, 3, 4, 5 and 6, and        form a ring with the carbon atom to which they are attached;    -   R⁶ is selected from —H and —C₁-C₈ alkyl;    -   R⁷ is selected from —H, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,        —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈        carbocycle), —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈        heterocycle);    -   each R⁸ is independently selected from —H, —OH, —C₁-C₈ alkyl,        —C₃-C₈ carbocycle and —O—(C₁-C₈ alkyl);    -   R⁹ is selected from —H and —C₁-C₈ alkyl;    -   X is —O—, —S—, —NH— or —N(R¹⁴)—;    -   R¹¹ is selected from —H, —OH, —NH₂, —NHR¹⁴, —N(R¹⁴)₂, —C₁-C₈        alkyl, —C₃-C₈ carbocycle, —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈        alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈ carbocycle), —C₃-C₈ heterocycle        and —C₁-C₈ alkyl-(C₃-C₈ heterocycle); or R¹¹ is an oxygen atom        which forms a carbonyl unit (C═O) with the carbon atom to which        it is attached and a hydrogen atom on this carbon atom is        replaced by one of the bonds in the (C═O) double bond;    -   each R¹² is independently selected from -aryl and —C₃-C₈        heterocycle;    -   each R¹⁴ is independently —H or —C₁-C₈ alkyl;    -   R¹⁶ is —Yy-Ww-A′        wherein    -   each —W— is independently an Amino Acid unit;    -   —Y— is a Spacer unit;    -   w is an integer ranging from 0 to 12;    -   y is 0, 1 or 2; and    -   -A′ is selected from

wherein

-   -   G is selected from —Cl, —Br, —I, —O-mesyl and —O-tosyl;    -   J is selected from —Cl, —Br, —I, —F, —OH, —O—N-succinimide,        —O-(4-nitrophenyl), —O-pentafluorophenyl, —O-tetrafluorophenyl        and —O—C(O)—OR¹⁸;    -   R¹⁷ is selected from —C₁-C₁₀ alkylene-, —C₃-C₈ carbocyclo-,        —O—(C₁-C₈ alkyl)-, -arylene-, —C₁-C₁₀ alkylene-arylene-,        -arylene-C₁-C₁₀ alkylene-, —C₁-C₁₀ alkylene-(C₃-C₈ carbocyclo)-,        —(C₃-C₈ carbocyclo)-C₁-C₁₀ alkylene-, —C₃-C₈ heterocyclo-,        —C₁-C₁₀ alkylene-(C₃-C₈ heterocyclo)-, —(C₃-C₈        heterocyclo)-C₁-C₁₀alkylene-, —(CH₂CH₂O)_(r)—, and        —(CH₂CH₂O)_(r)—CH₂—; r is an integer ranging from 1-10; and    -   R¹⁸ is —C₁-C₈ alkyl or -aryl.

In another aspect, the present invention provides compounds of theformula IId:

-   -   and pharmaceutically acceptable salts and solvates thereof    -   wherein, independently at each location:    -   R¹ is selected from —H, —C₁-C₈ alkyl and —C₃-C₈ carbocycle; and        R² is selected from —H and —C₁-C₈ alkyl; or R¹ and R² join, have        the formula —(CR^(a)R^(b))_(n)— wherein R^(a) and R^(b) are        independently selected from —H, —C₁-C₈ alkyl and —C₃-C₈        carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a        ring with the nitrogen atom to which they are attached;    -   R³ is selected from —H, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,        —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈        carbocycle), —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈        heterocycle);    -   R⁴ is selected from —H, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,        —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈        carbocycle), —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈        heterocycle) wherein R⁵ is selected from —H and -methyl; or R⁴        and R⁵ join, have the formula —(CR^(a)R^(b))_(n)— wherein R^(a)        and R^(b) are independently selected from —H, —C₁-C₈ alkyl and        —C₃-C₈ carbocycle and n is selected from 2, 3, 4, 5 and 6, and        form a ring with the carbon atom to which they are attached;    -   R⁶ is selected from —H and —C₁-C₈ alkyl;    -   R⁷ is selected from —H, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,        —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈        carbocycle), —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈        heterocycle);    -   each R⁸ is independently selected from —H, —OH, —C₁-C₈ alkyl,        —C₃-C₈ carbocycle and —O—(C₁-C₈ alkyl);    -   R⁹ is selected from —H and —C₁-C₈ alkyl;    -   X is —O—, —S—, —NH— or —N(R¹⁴)—;    -   R¹¹ is selected from —H, —OH, —NH₂, —NHR¹⁴, —N(R¹⁴)₂, —C₁-C₈        alkyl, —C₃-C₈ carbocycle, —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈        alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈ carbocycle), —C₃-C₈ heterocycle        and —C₁-C₈ alkyl-(C₃-C₈ heterocycle); or R¹¹ is an oxygen atom        which forms a carbonyl unit (C═O) with the carbon atom to which        it is attached and a hydrogen atom on this carbon atom is        replaced by one of the bonds in the (C═O) double bond;    -   each R¹² is independently selected from -aryl and —C₃-C₈        heterocycle;    -   each R¹⁴ is independently —H or —C₁-C₈ alkyl;    -   R¹⁵ is -arylene-, —C₃-C₈ carbocyclo- or —C₃-C₈ heterocyclo-;    -   R¹⁶ is —Yy-Ww-A′        wherein    -   each —W— is independently an Amino Acid unit;    -   —Y— is a Spacer unit;    -   w is an integer ranging from 0 to 12;    -   y is 0, 1 or 2; and    -   -A′ is selected from

wherein

-   -   G is selected from —Cl, —Br, —I, —O-mesyl and —O-tosyl;    -   J is selected from —Cl, —Br, —I, —F, —OH, —O—N-succinimide,        —O-(4-nitrophenyl), —O-pentafluorophenyl, —O-tetrafluorophenyl        and —O—C(O)—OR¹⁸;    -   R¹⁷ is selected from —C₁-C₁₀ alkylene-, —C₃-C₈ carbocyclo-,        —O—(C₁-C₈ alkyl)-, -arylene-, —C₁-C₁₀ alkylene-arylene-,        -arylene-C₁-C₁₀ alkylene-, —C₁-C₁₀ alkylene-(C₃-C₈ carbocyclo)-,        —(C₃-C₈ carbocyclo)-C₁-C₁₀ alkylene-, —C₃-C₈ heterocyclo-,        —C₁-C₁₀ alkylene-(C₃-C₈ heterocyclo)-, —(C₃-C₈        heterocyclo)-C₁-C₁₀ alkylene-, —(CH₂CH₂O)_(r)—, and        —(CH₂CH₂O)_(r)—CH₂—; r is an integer ranging from 1-10; and    -   R¹⁸ is —C₁-C₈ alkyl or -aryl.

In another aspect, the present invention provides compounds of theformula IIe:

-   -   and pharmaceutically acceptable salts and solvates thereof    -   wherein, independently at each location:    -   R¹ is selected from —H, —C₁-C₈ alkyl and —C₃-C₈ carbocycle; and        R² is selected from —H and —C₁-C₈ alkyl; or R¹ and R² join, have        the formula —(CR^(a)R^(b))_(n)— wherein R^(a) and R^(b) are        independently selected from —H, —C₁-C₈ alkyl and —C₃-C₈        carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a        ring with the nitrogen atom to which they are attached;    -   R³ is selected from —H, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,        —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈        carbocycle), —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈        heterocycle);    -   R⁴ is selected from —H, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,        —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈        carbocycle), —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈        heterocycle) wherein R⁵ is selected from —H and -methyl; or R⁴        and R⁵ join, have the formula —(CR^(a)R^(b))_(n)— wherein R^(a)        and R^(b) are independently selected from —H, —C₁-C₈ alkyl and        —C₃-C₈ carbocycle and n is selected from 2, 3, 4, 5 and 6, and        form a ring with the carbon atom to which they are attached;    -   R⁶ is selected from —H and —C₁-C₈ alkyl;    -   R⁷ is selected from —H, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,        —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈        carbocycle), —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈        heterocycle);    -   each R⁸ is independently selected from —H, —OH, —C₁-C₈ alkyl,        —C₃-C₈ carbocycle and —O—(C₁-C₈ alkyl);    -   R⁹ is selected from —H and —C₁-C₈ alkyl;    -   X is —O—, —S—, —NH— or —N(R¹⁴)—;    -   Z is —O—, —S—, —NH— or —N(R¹⁴)—;    -   R¹¹ is selected from —H, —OH, —NH₂, —NHR¹⁴, —N(R¹⁴)₂, —C₁-C₈        alkyl, —C₃-C₈ carbocycle, —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈        alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈ carbocycle), —C₃-C₈ heterocycle        and —C₁-C₈ alkyl-(C₃-C₈ heterocycle); or R¹¹ is an oxygen atom        which forms a carbonyl unit (C═O) with the carbon atom to which        it is attached and a hydrogen atom on this carbon atom is        replaced by one of the bonds in the (C═O) double bond;    -   each R¹² is independently selected from -aryl and —C₃-C₈        heterocycle;    -   each R¹⁴ is independently —H or —C₁-C₈ alkyl;    -   R¹⁵ is -arylene-, —C₃-C₈ carbocyclo- or —C₃-C₈ heterocyclo-;    -   R¹⁶ is —Yy-Ww-A′        wherein    -   each —W— is independently an Amino Acid unit;    -   —Y— is a Spacer unit;    -   w is an integer ranging from 0 to 12;    -   y is 0, 1 or 2; and    -   -A′ is selected from

wherein

-   -   G is selected from —Cl, —Br, —I, —O-mesyl and —O-tosyl;    -   J is selected from —Cl, —Br, —I, —F, —OH, —O—N-succinimide,        —O-(4-nitrophenyl), —O-pentafluorophenyl, —O-tetrafluorophenyl        and —O—C(O)—OR¹⁸;    -   R¹⁷ is selected from —C₁-C₁₀ alkylene-, —C₃-C₈ carbocyclo-,        —O—(C₁-C₈ alkyl)-, -arylene-, —C₁-C₁₀ alkylene-arylene-,        -arylene-C₁-C₁₀ alkylene-, —C₁-C₁₀ alkylene-(C₃-C₈ carbocyclo)-,        —(C₃-C₈ carbocyclo)-C₁-C₁₀ alkylene-, —C₃-C₈ heterocyclo-,        —C₁-C₁₀ alkylene-(C₃-C₈ heterocyclo)-, —(C₃-C₈        heterocyclo)-C₁-C₁₀alkylene-, —(CH₂CH₂O)_(r)—, and        —(CH₂CH₂O)_(r)—CH₂—; r is an integer ranging from 1-10; and    -   R¹⁸ is —C₁-C₈ alkyl or -aryl.

In another aspect, the present invention provides compounds of theformula IIf:

-   -   and pharmaceutically acceptable salts and solvates thereof    -   wherein, independently at each location:    -   R¹ is selected from —H, —C₁-C₈ alkyl and —C₃-C₈ carbocycle; and        R² is selected from —H and —C₁-C₈ alkyl; or R¹ and R² join, have        the formula —(CR^(a)R^(b))_(n)— wherein R^(a) and R^(b) are        independently selected from —H, —C₁-C₈ alkyl and —C₃-C₈        carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a        ring with the nitrogen atom to which they are attached;    -   R³ is selected from —H, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,        —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈        carbocycle), —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈        heterocycle);    -   R⁴ is selected from —H, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,        —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈        carbocycle), —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈        heterocycle) wherein R⁵ is selected from —H and -methyl; or R⁴        and R⁵ join, have the formula —(CR^(a)R^(b))_(n)— wherein R^(a)        and R^(b) are independently selected from —H, —C₁-C₈ alkyl and        —C₃-C₈ carbocycle and n is selected from 2, 3, 4, 5 and 6, and        form a ring with the carbon atom to which they are attached;    -   R⁶ is selected from —H and —C₁-C₈ alkyl;    -   R⁷ is selected from —H, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,        —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈        carbocycle), —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈        heterocycle);    -   each R⁸ is independently selected from —H, —OH, —C₁-C₈ alkyl,        —C₃-C₈ carbocycle and —O—(C₁-C₈ alkyl);    -   R⁹ is selected from —H and —C₁-C₈ alkyl;    -   X is —O—, —S—, —NH— or —N(R¹⁴)—;    -   Z is —O—, —S—, —NH— or —N(R¹⁴)—;    -   R¹¹ is selected from —H, —OH, —NH₂, —NHR¹⁴, —N(R¹⁴)₂, —C₁-C₈        alkyl, —C₃-C₈ carbocycle, —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈        alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈ carbocycle), —C₃-C₈ heterocycle        and —C₁-C₈ alkyl-(C₃-C₈ heterocycle); or R¹¹ is an oxygen atom        which forms a carbonyl unit (C═O) with the carbon atom to which        it is attached and a hydrogen atom on this carbon atom is        replaced by one of the bonds in the (C═O) double bond;    -   each R¹² is independently selected from -aryl and —C₃-C₈        heterocycle;    -   each R¹⁴ is independently —H or —C₁-C₈ alkyl;    -   R¹⁵ is -arylene-, —C₃-C₈ carbocyclo- or —C₃-C₈ heterocyclo-;    -   R¹⁶ is —Yy-Ww-A′        wherein    -   each —W— is independently an Amino Acid unit;    -   —Y— is a Spacer unit;    -   w is an integer ranging from 0 to 12;    -   y is 0, 1 or 2; and    -   -A′ is selected from

wherein

-   -   G is selected from —Cl, —Br, —I, —O-mesyl and —O-tosyl;    -   J is selected from —Cl, —Br, —I, —F, —OH, —O—N-succinimide,        —O-(4-nitrophenyl), —O-pentafluorophenyl, —O-tetrafluorophenyl        and —O—C(O)—OR¹⁸;    -   R¹⁷ is selected from —C₁-C₁₀ alkylene-, —C₃-C₈ carbocyclo-,        —O—(C₁-C₈ alkyl)-, -arylene-, —C₁-C₁₀ alkylene-arylene-,        -arylene-C₁-C₁₀ alkylene-, —C₁-C₁₀ alkylene-(C₃-C₈ carbocyclo)-,        —(C₃-C₈ carbocyclo)-C₁-C₁₀ alkylene-, —C₃-C₈ heterocyclo-,        —C₁-C₁₀ alkylene-(C₃-C₈ heterocyclo)-, —(C₃-C₈        heterocyclo)-C₁-C₁₀alkylene-, —(CH₂CH₂O)_(r)—, and        —(CH₂CH₂O)_(r)—CH₂—; r is an integer ranging from 1-10; and    -   R¹⁸ is —C₁-C₈ alkyl or -aryl.

In one embodiment R¹ is selected from —C₁-C₈ alkyl and —C₃-C₈carbocycle; and R² is selected from —H and —C₁-C₈ alkyl; or R¹ and R²join, have the formula —(CR^(a)R^(b))_(n)— wherein R^(a) and R^(b) areindependently selected from —H, —C₁-C₈ alkyl and —C₃-C₈ carbocycle and nis selected from 2, 3, 4, 5 and 6, and form a ring with the nitrogenatom to which they are attached

Illustrative compounds of formula IIf have the structure:

and pharmaceutically acceptable salts and solvates thereof

In another aspect, the present invention provides compounds of theformula IIg:

-   -   and pharmaceutically acceptable salts and solvates thereof    -   wherein, independently at each location:    -   R² is selected from —H and —C₁-C₈ alkyl;    -   R³ is selected from —H, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,        —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈        carbocycle), —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈        heterocycle);    -   R⁴ is selected from —H, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,        —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈        carbocycle), —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈        heterocycle) wherein R⁵ is selected from —H and -methyl; or R⁴        and R⁵ join, have the formula —(CR^(a)R^(b))_(n)— wherein R^(a)        and R^(b) are independently selected from —H, —C₁-C₈ alkyl and        —C₃-C₈ carbocycle and n is selected from 2, 3, 4, 5 and 6, and        form a ring with the carbon atom to which they are attached;    -   R⁶ is selected from —H and —C₁-C₈ alkyl;    -   R⁷ is selected from —H, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,        —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈        carbocycle), —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈        heterocycle);    -   each R⁸ is independently selected from —H, —OH, —C₁-C₈ alkyl,        —C₃-C₈ carbocycle and —O—(C₁-C₈ alkyl);    -   R⁹ is selected from —H and —C₁-C₈ alkyl;    -   Z is —O—, —S—, —NH— or —N(R¹⁴)—;    -   R¹¹ is selected from —H, —OH, —NH₂, —NHR¹⁴, —N(R¹⁴)₂, —C₁-C₈        alkyl, —C₃-C₈ carbocycle, —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈        alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈ carbocycle), —C₃-C₈ heterocycle        and —C₁-C₈ alkyl-(C₃-C₈ heterocycle); or R¹¹ is an oxygen atom        which forms a carbonyl unit (C═O) with the carbon atom to which        it is attached and a hydrogen atom on this carbon atom is        replaced by one of the bonds in the (C═O) double bond;    -   each R¹² is independently selected from -aryl and —C₃-C₈        heterocycle;    -   each R¹⁴ is independently —H or —C₁-C₈ alkyl;    -   R¹⁶ is —Yy-Ww-A′        wherein    -   each —W— is independently an Amino Acid unit;    -   —Y— is a Spacer unit;    -   w is an integer ranging from 0 to 12;    -   y is 0, 1 or 2; and    -   -A′ is selected from

wherein

-   -   G is selected from —Cl, —Br, —I, —O-mesyl and —O-tosyl;    -   J is selected from —Cl, —Br, —I, —F, —OH, —O—N-succinimide,        —O-(4-nitrophenyl), —O-pentafluorophenyl, —O-tetrafluorophenyl        and —O—C(O)—OR¹⁸;    -   R¹⁷ is selected from —C₁-C₁₀ alkylene-, —C₃-C₈ carbocyclo-,        —O—(C₁-C₈ alkyl)-, -arylene-, —C₁-C₁₀ alkylene-arylene-,        -arylene-C₁-C₁₀ alkylene-, —C₁-C₁₀ alkylene-(C₃-C₈ carbocyclo)-,        —(C₃-C₈ carbocyclo)-C₁-C₁₀ alkylene-, —C₃-C₈ heterocyclo-,        —C₁-C₁₀ alkylene-(C₃-C₈ heterocyclo)-, —(C₃-C₈        heterocyclo)-C₁-C₁₀alkylene-, —(CH₂CH₂O)_(r)—, and        —(CH₂CH₂O)_(r)—CH₂—; r is an integer ranging from 1-10; and    -   R¹⁸ is —C₁-C₈ alkyl or -aryl.

In another aspect, the present invention provides compounds of theformula IIh:

-   -   and pharmaceutically acceptable salts and solvates thereof    -   wherein, independently at each location:    -   R² is selected from —H and —C₁-C₈ alkyl;    -   R³ is selected from —H, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,        —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈        carbocycle), —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈        heterocycle);    -   R⁴ is selected from —H, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,        —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈        carbocycle), —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈        heterocycle) wherein R⁵ is selected from —H and -methyl; or R⁴        and R⁵ join, have the formula —(CR^(a)R^(b))_(n)— wherein R^(a)        and R^(b) are independently selected from —H, —C₁-C₈ alkyl and        —C₃-C₈ carbocycle and n is selected from 2, 3, 4, 5 and 6, and        form a ring with the carbon atom to which they are attached;    -   R⁶ is selected from —H and —C₁-C₈ alkyl;    -   R⁷ is selected from —H, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,        —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈        carbocycle), —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈        heterocycle);    -   each R⁸ is independently selected from —H, —OH, —C₁-C₈ alkyl,        —C₃-C₈ carbocycle and —O—(C₁-C₈ alkyl);    -   R⁹ is selected from —H and —C₁-C₈ alkyl;    -   Z is —O—, —S—, —NH— or —N(R¹⁴)—;    -   R¹¹ is selected from —H, —OH, —NH₂, —NHR¹⁴, —N(R¹⁴)₂, —C₁-C₈        alkyl, —C₃-C₈ carbocycle, —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈        alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈ carbocycle), —C₃-C₈ heterocycle        and —C₁-C₈ alkyl-(C₃-C₈ heterocycle); or R¹¹ is an oxygen atom        which forms a carbonyl unit (C═O) with the carbon atom to which        it is attached and a hydrogen atom on this carbon atom is        replaced by one of the bonds in the (C═O) double bond;    -   each R¹² is independently selected from -aryl and —C₃-C₈        heterocycle;    -   each R¹⁴ is independently —H or —C₁-C₈ alkyl;    -   R¹⁶ is —Yy-Ww-A′        wherein    -   each —W— is independently an Amino Acid unit;    -   —Y— is a Spacer unit;    -   w is an integer ranging from 0 to 12;    -   y is 0, 1 or 2; and    -   -A′ is selected from

wherein

-   -   G is selected from —Cl, —Br, —I, —O-mesyl and —O-tosyl;    -   J is selected from —Cl, —Br, —I, —F, —OH, —O—N-succinimide,        —O-(4-nitrophenyl), —O-pentafluorophenyl, —O-tetrafluorophenyl        and —O—C(O)—OR¹⁸;    -   R¹⁷ is selected from —C₁-C₁₀ alkylene-, —C₃-C₈ carbocyclo-,        —O—(C₁-C₈ alkyl)-, -arylene-, —C₁-C₁₀ alkylene-arylene-,        -arylene-C₁-C₁₀ alkylene-, —C₁-C₁₀ alkylene-(C₃-C₈ carbocyclo)-,        —(C₃-C₈ carbocyclo)-C₁-C₁₀ alkylene-, —C₃-C₈ heterocyclo-,        —C₁-C₁₀ alkylene-(C₃-C₈ heterocyclo)-, —(C₃-C₈        heterocyclo)-C₁-C₁₀ alkylene-, —(CH₂CH₂O)_(r)—, and        —(CH₂CH₂O)_(r)—CH₂—; r is an integer ranging from 1-10; and    -   R¹⁸ is —C₁-C₈ alkyl or -aryl.

In another aspect, the present invention provides compounds of theformula IIi:

-   -   and pharmaceutically acceptable salts and solvates thereof    -   wherein, independently at each location:    -   R² is selected from —H and —C₁-C₈ alkyl;    -   R³ is selected from —H, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,        —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈        carbocycle), —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈        heterocycle);    -   R⁴ is selected from —H, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,        —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈        carbocycle), —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈        heterocycle) wherein R⁵ is selected from —H and -methyl; or R⁴        and R⁵ join, have the formula —(CR^(a)R^(b))_(n)— wherein R^(a)        and R^(b) are independently selected from —H, —C₁-C₈ alkyl and        —C₃-C₈ carbocycle and n is selected from 2, 3, 4, 5 and 6, and        form a ring with the carbon atom to which they are attached;    -   R⁶ is selected from —H and —C₁-C₈ alkyl;    -   R⁷ is selected from —H, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,        —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈        carbocycle), —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈        heterocycle);    -   each R⁸ is independently selected from —H, —OH, —C₁-C₈ alkyl,        —C₃-C₈ carbocycle and —O—(C₁-C₈ alkyl);    -   R⁹ is selected from —H and —C₁-C₈ alkyl;    -   R¹¹ is selected from —H, —OH, —NH₂, —NHR¹⁴, —N(R¹⁴)₂, —C₁-C₈        alkyl, —C₃-C₈ carbocycle, —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈        alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈ carbocycle), —C₃-C₈ heterocycle        and —C₁-C₈ alkyl-(C₃-C₈ heterocycle); or R¹¹ is an oxygen atom        which forms a carbonyl unit (C═O) with the carbon atom to which        it is attached and a hydrogen atom on this carbon atom is        replaced by one of the bonds in the (C═O) double bond;    -   each R¹² is independently selected from -aryl and —C₃-C₈        heterocycle;    -   R¹³ is selected from hydrogen, —OH, —NH₂, —NHR¹⁴, —N(R¹⁴)₂,        C₁-C₈ alkyl, C₃-C₈ carbocycle, —O—(C₁-C₈ alkyl), -aryl,        alkyl-aryl, alkyl-(C₃-C₈ carbocycle), C₃-C₈ heterocycle and        alkyl-(C₃-C₈ heterocycle);    -   each R¹⁴ is independently —H or —C₁-C₈ alkyl;    -   R¹⁶ is —Yy-Ww-A′        wherein    -   each —W— is independently an Amino Acid unit;    -   —Y— is a Spacer unit;    -   w is an integer ranging from 0 to 12;    -   y is 0, 1 or 2; and    -   -A′ is selected from

wherein

-   -   G is selected from —Cl, —Br, —I, —O-mesyl and —O-tosyl;    -   J is selected from —Cl, —Br, —I, —F, —OH, —O—N-succinimide,        —O-(4-nitrophenyl), —O-pentafluorophenyl, —O-tetrafluorophenyl        and —O—C(O)—OR¹⁸;    -   R¹⁷ is selected from —C₁-C₁₀ alkylene-, —C₃-C₈ carbocyclo-,        —O—(C₁-C₈ alkyl)-, -arylene-, —C₁-C₁₀ alkylene-arylene-,        -arylene-C₁-C₁₀ alkylene-, —C₁-C₁₀ alkylene-(C₃-C₈ carbocyclo)-,        —(C₃-C₈ carbocyclo)-C₁-C₁₀ alkylene-, —C₃-C₈ heterocyclo-,        —C₁-C₁₀ alkylene-(C₃-C₈ heterocyclo)-, —(C₃-C₈        heterocyclo)-C₁-C₁₀alkylene-, —(CH₂CH₂O)_(r)—, and        —(CH₂CH₂O)_(r)—CH₂—; r is an integer ranging from 1-10; and    -   R¹⁸ is —C₁-C₈ alkyl or -aryl.

Illustrative compounds of formula IIi have the structures:

and pharmaceutically acceptable salts and solvates thereof

The compounds of formulas IIa-i are useful for treating or preventingcancer, an autoimmune disease or an infectious disease in an animal.

The Limit Unit

The Linker unit of the Drug-Linker-Ligand Conjugate links the Drug unitand the Ligand unit and has the formula:

-A_(a)-W_(w)—Y_(y)—

wherein:

-   -   -A- is a Stretcher unit;    -   a is 0 or 1;    -   each —W— is independently an Amino Acid unit;    -   w is independently an integer ranging from 0 to 12;    -   —Y— is a Spacer unit; and    -   y is 0, 1 or 2.

The Stretcher Unit

The Stretcher unit (-A-), when present, links a Ligand unit to an aminoacid unit (—W—). In this regard a Ligand (L) has a functional group thatcan form a bond with a functional group of a Stretcher. Usefulfunctional groups that can be present on a ligand, either naturally orvia chemical manipulation include, but are not limited to, sulfhydryl(—SH), amino, hydroxyl, carboxy, the anomeric hydroxyl group of acarbohydrate, and carboxyl. Preferred Ligand functional groups aresulfhydryl and amino. Sulfhydryl groups can be generated by reduction ofan intramolecular disulfide bond of a Ligand. Alternatively, sulfhydrylgroups can be generated by reaction of an amino group of a lysine moietyof a Ligand using 2-iminothiolane (Traut's reagent) or anothersulfhydryl generating reagent.

In one embodiment, the Stretcher unit forms a bond with a sulfur atom ofthe Ligand unit. The sulfur atom can be derived from a sulfhydryl groupof a Ligand. Representative Stretcher units of this embodiment aredepicted within the square brackets of Formulas (IIIa) and (IIIb),wherein L-, —W—, —Y—, -D, w and y are as defined above and R¹⁷ isselected from —C₁-C₁₀ alkylene-, —C₃-C₈ carbocyclo-, —O—(C₁-C₈ alkyl)-,-arylene-, —C₁-C₁₀ alkylene-arylene-, -arylene-C₁-C₁₀ alkylene-, —C₁-C₁₀alkylene-(C₃-C₈ carbocyclo)-, —(C₃-C₈ carbocyclo)-C₁-C₁₀ alkylene-,—C₃-C₈ heterocyclo-, —C₁-C₁₀ alkylene-(C₃-C₈ heterocyclo)-, —(C₃-C₈heterocyclo)-C₁-C₁₀ alkylene-, —(CH₂CH₂O)_(r)—, and —(CH₂CH₂O)_(r)—CH₂—;and r is an integer ranging from 1-10.

An illustrative Stretcher unit is that of formula (IIIa) where R¹⁷ is—(CH₂)₅—:

Another illustrative Stretcher unit is that of formula (IIIa) where R¹⁷is —(CH₂CH₂O)_(r)—CH₂—; and r is 2:

Still another illustrative Stretcher unit is that of formula (IIIb)where R¹⁷ is —(CH₂)₅—:

In another embodiment, the Stretcher unit is linked to the Ligand unitvia a disulfide bond between a sulfur atom of the Ligand unit and asulfur atom of the Stretcher unit. A representative Stretcher unit ofthis embodiment is depicted within the square brackets of Formula (IV),wherein R¹⁷, L-, —W—, —Y—, -D, w and y are as defined above.

In yet another embodiment, the reactive group of the Stretcher containsa reactive site that can form a bond with a primary or secondary aminogroup of a Ligand. Example of these reactive sites include, but are notlimited to, activated esters such as succinimide esters, 4-nitrophenylesters, pentafluorophenyl esters, tetrafluorophenyl esters, anhydrides,acid chlorides, sulfonyl chlorides, isocyanates and isothiocyanates.Representative Stretcher units of this embodiment are depicted withinthe square brackets of Formulas (Va) and (Vb), wherein —R¹⁷—, L-, —W—,—Y—, -D, w and y are as defined above;

In yet another aspect of the invention, the reactive group of theStretcher contains a reactive site that is reactive to a carbohydrate's(—CHO) group that can be present on a Ligand. For example, acarbohydrate can be mildly oxidized using a reagent such as sodiumperiodate and the resulting (—CHO) unit of the oxidized carbohydrate canbe condensed with a Stretcher that contains a functionality such as ahydrazide, an oxime, a primary or secondary amine, a hydrazine, athiosemicarbazone, a hydrazine carboxylate, and an arylhydrazide such asthose described by Kaneko, T. et al. Bioconjugate Chem 1991, 2, 133-41.Representative Stretcher units of this embodiment are depicted withinthe square brackets of Formulas (VIa)-(VIc), wherein —R¹⁷—, L-, —W—,—Y—, -D, w and y are as defined above.

The Amino Acid Unit

The Amino Acid unit (—W—), when present, links the Stretcher unit to theSpacer unit if the Spacer unit is present, links the Stretcher unit tothe Drug unit if the Spacer unit is absent, and links the Ligand unit tothe Drug unit if the Stretcher unit and Spacer unit are absent.

—W_(w)— is a dipeptide, tripeptide, tetrapeptide, pentapeptide,hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide,undecapeptide or dodecapeptide unit. Each —W— unit independently has theformula denoted below in the square brackets, and w is an integerranging from 0 to 12:

wherein R¹⁹ is hydrogen, methyl, isopropyl, isobutyl, sec-butyl, benzyl,p-hydroxybenzyl, —CH₂OH, —CH(OH)CH₃, —CH₂CH₂SCH₃, —CH₂CONH₂, —CH₂COOH,—CH₂CH₂CONH₂, —CH₂CH₂COOH, —(CH₂)₃NHC(═NH)NH₂, —(CH₂)₃NH₂,—(CH₂)₃NHCOCH₃, —(CH₂)₃NHCHO, —(CH₂)₄NHC(═NH)NH₂, —(CH₂)₄NH₂,—(CH₂)₄NHCOCH₃, —(CH₂)₄NHCHO, —(CH₂)₃NHCONH₂, —(CH₂)₄NHCONH₂,—CH₂CH₂CH(OH)CH₂NH₂, 2-pyridylmethyl-, 3-pyridylmethyl-,4-pyridylmethyl-, phenyl, cyclohexyl,

The Amino Acid unit of the Compounds of the Invention can beenzymatically cleaved by one or more enzymes, including atumor-associated protease, to liberate the Drug unit (-D), which in oneembodiment is protonated in vivo upon release to provide a Drug (D).

Illustrative W_(w) units are represented by formulas (VII)-(IX):

wherein R²⁰ and R²¹ are as follows:

R²⁰ R²¹ benzyl (CH₂)₄NH₂; methyl (CH₂)₄NH₂; isopropyl (CH₂)₄NH₂;isopropyl (CH₂)₃NHCONH₂; benzyl (CH₂)₃NHCONH₂; isobutyl (CH₂)₃NHCONH₂;sec-butyl (CH₂)₃NHCONH₂;

(CH₂)₃NHCONH₂; benzyl methyl; and benzyl (CH₂)₃NHC(═NH)NH₂;

wherein R²⁰, R²¹ and R²² are as follows:

R²⁰ R²¹ R²² benzyl benzyl (CH₂)₄NH₂; isopropyl benzyl (CH₂)₄NH₂; and Hbenzyl (CH₂)₄NH₂;

wherein R²⁰, R²¹, R²² and R²³ are as follows:

R²⁰ R²¹ R²² R²³ H benzyl isobutyl H; and methyl isobutyl methylisobutyl.

Preferred Amino Acid units include, but are not limited to, units offormula (VII) where: R²⁰ is benzyl and R²¹ is —(CH₂)₄NH₂, R²⁰ isopropyland R²¹ is —(CH₂)₄NH₂; R²⁰ isopropyl and R²¹ is —(CH₂)₃NHCONH₂. Anotherpreferred Amino Acid unit is a unit of formula (VIII) where R²⁰ isbenzyl, R²¹ is benzyl, and R²² is —(CH₂)₄NH₂.

—W_(w)— units useful in the present invention can be designed andoptimized in their selectivity for enzymatic cleavage by a particularenzymes, for example, a tumor-associated protease. In one embodiment, a—W_(w)— unit is that whose cleavage is catalyzed by cathepsin B, C andD, or a plasmin protease.

In one embodiment, —W_(w)— is a dipeptide, tripeptide or pentapeptide.

Where R¹⁹, R²⁰, R²¹, R²² or R²³ is other than hydrogen, the carbon atomto which R¹⁹, R²⁰, R²¹, R²² or R₂₃ is attached is chiral.

Each carbon atom to which R¹⁹, R²⁰, R²¹, R²² or R²³ is attached isindependently in the (S) or (R) configuration.

The Spacer Unit

The Spacer unit (—Y—), when present, links an Amino Acid unit to theDrug unit when an Amino Acid unit is present. Alternately, the Spacerunit links the Stretcher unit to the Drug unit when the Amino Acid unitis absent. The Spacer unit also links the Drug unit to the ligand unitwhen both the Amino Acid unit and Stretcher unit are absent. Spacerunits are of two general types: self-immolative and non self-immolative.A non self-immolative Spacer unit is one in which part or all of theSpacer unit remains bound to the Drug unit after cleavage, particularlyenzymatic, of an Amino Acid unit from the Drug-Linker-Ligand Conjugateor the Drug-Linker Compound. Examples of a non self-immolative Spacerunit include, but are not limited to a (glycine-glycine) Spacer unit anda glycine Spacer unit (both depicted in Scheme 1). When a Compound ofthe Invention containing a glycine-glycine Spacer unit or a glycineSpacer unit undergoes enzymatic cleavage via a tumor-cellassociated-protease, a cancer-cell-associated protease or alymphocyte-associated protease, a glycine-glycine-Drug moiety or aglycine-Drug moiety is cleaved from L-A_(a)-W_(w)—. In one embodiment,an independent hydrolysis reaction takes place within the target cell,cleaving the glycine-Drug unit bond and liberating the Drug.

In a preferred embodiment, —Y_(y)— is a p-aminobenzyl alcohol (PAB) unit(see Schemes 2 and 3) whose phenylene portion is substituted with Q_(m)where Q is is —C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -halogen, -nitro or-cyano; and m is an integer ranging from 0-4.

In one embodiment, a non self-immolative Spacer unit (—Y—) is -Gly-Gly-.

In another embodiment, a non self-immolative the Spacer unit (—Y—) is-Gly-.

In one embodiment, the invention provides a Drug-Linker Compound or aDrug-Linker Ligand Conjugate in which the Spacer unit is absent (y=0),or a pharmaceutically acceptable salt or solvate thereof.

Alternatively, a Compound of the Invention containing a self-immolativeSpacer unit can release -D without the need for a separate hydrolysisstep. In this embodiment, —Y— is a PAB group that is linked to —W_(w)—via the amino nitrogen atom of the PAB group, and connected directly to-D via a carbonate, carbamate or ether group. Without being bound bytheory, Scheme 2 depicts a possible mechanism of Drug release of a PABgroup which is attached directly to -D via a carbamate or carbonategroup.

where Q is —C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -halogen, -nitro or -cyano; mis an integer ranging from 0-4; and p ranges from 1 to about 20.

Without being bound by theory, Scheme 3 depicts a possible mechanism ofDrug release of a PAB group which is attached directly to -D via anether or amine linkage.

where Q is —C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -halogen, -nitro or -cyano; mis an integer ranging from 0-4; and p ranges from 1 to about 20.

Other examples of self-immolative spacers include, but are not limitedto, aromatic compounds that are electronically similar to the PAB groupsuch as 2-aminoimidazol-5-methanol derivatives (see Hay et al., Bioorg.Med. Chem. Lett., 1999, 9, 2237) and ortho or para-aminobenzylacetals.Spacers can be used that undergo cyclization upon amide bond hydrolysis,such as substituted and unsubstituted 4-aminobutyric acid amides(Rodrigues et al., Chemistry Biology, 1995, 2, 223), appropriatelysubstituted bicyclo[2.2.1] and bicyclo[2.2.2] ring systems (Storm, etal., J. Amer. Chem. Soc., 1972, 94, 5815) and 2-aminophenylpropionicacid amides (Amsberry, et al., J. Org. Chem., 1990, 55, 5867).Elimination of amine-containing drugs that are substituted at thea-position of glycine (Kingsbury, et al., J. Med. Chem., 1984, 27, 1447)are also examples of self-immolative spacer useful in the Compounds ofthe Invention.

In a preferred embodiment, the Spacer unit is a branchedbis(hydroxymethyl)styrene (BHMS) unit as depicted in Scheme 4, which canbe used to incorporate and release multiple drugs.

where Q is —C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -halogen, -nitro or -cyano; mis an integer ranging from 0-4; n is 0 or 1; and p ranges raging from 1to about 20.

In one embodiment, the -D moieties are the same.

In another embodiment, the -D moieties are different.

Preferred Spacer units (—Y_(y)—) are represented by Formulas (X)-(XII):

where Q is —C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -halogen, -nitro or -cyano;and m is an integer ranging from 0-4;

The Drug Unit

-D is a Drug unit having a nitrogen or oxygen atom that can form a bondwith the Spacer unit when y=1 or 2 or with the C-terminal carbonyl groupof an Amino Acid unit when y=0.

In one embodiment, -D is represented by the formula:

-   -   wherein, independently at each location:    -   R² is selected from —H and —C₁-C₈ alkyl;    -   R³ is selected from —H, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,        —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈        carbocycle), —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈        heterocycle);    -   R⁴ is selected from —H, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,        —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈        carbocycle), —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈        heterocycle) wherein R⁵ is selected from —H and -methyl; or R⁴        and R⁵ join, have the formula —(CR^(a)R^(b))_(n)— wherein R^(a)        and R^(b) are independently selected from —H, —C₁-C₈ alkyl and        —C₃-C₈ carbocycle and n is selected from 2, 3, 4, 5 and 6, and        form a ring with the carbon atom to which they are attached;    -   R⁶ is selected from —H and —C₁-C₈ alkyl;    -   R⁷ is selected from —H, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,        —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈        carbocycle), —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈        heterocycle);    -   each R⁸ is independently selected from —H, —OH, —C₁-C₈ alkyl,        —C₃-C₈ carbocycle and —O—(C₁-C₈ alkyl);    -   R⁹ is selected from —H and —C₁-C₈ alkyl;    -   R¹⁰ is selected from

-   -   Z is —O—, —S—, —NH— or —N(R¹⁴)—;    -   R¹¹ is selected from —H, —OH, —NH₂, —NHR¹⁴, —N(R¹⁴)₂, —C₁-C₈        alkyl, —C₃-C₈ carbocycle, —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈        alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈ carbocycle), —C₃-C₈ heterocycle        and —C₁-C₈ alkyl-(C₃-C₈ heterocycle); or R¹¹ is an oxygen atom        which forms a carbonyl unit (C═O) with the carbon atom to which        it is attached and a hydrogen atom on this carbon atom is        replaced by one of the bonds in the (C═O) double bond;    -   each R¹² is independently selected from -aryl and —C₃-C₈        heterocycle;    -   R¹³ is selected from —H, —OH, —NH₂, —NHR¹⁴, —N(R¹⁴)₂, —C₁-C₈        alkyl, —C₃-C₈ carbocycle, —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈        alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈ carbocycle), C₃-C₈ heterocycle        and —C₁-C₈ alkyl-(C₃-C₈ heterocycle); and    -   each R¹⁴ is independently —H or —C₁-C₈ alkyl.

In one embodiment, R¹⁰ is selected from

In a preferred embodiment, -D has the formula

-   -   or a pharmaceutically acceptable salt or solvate thereof,    -   wherein, independently at each location:    -   R² is selected from —H and -methyl;    -   R³ is selected from —H, -methyl, and -isopropyl;    -   R⁴ is selected from —H and -methyl; R⁵ is selected from        -isopropyl, -isobutyl, -sec-butyl, -methyl and -t-butyl; or R⁴        and R⁵ join, have the formula —(CR^(a)R^(b))_(n)— where R^(a)        and R^(b) are independently selected from —H, —C₁-C₈ alkyl, and        —C₃-C₈ carbocycle, and n is selected from 2, 3, 4, 5 and 6, and        form a ring with the carbon atom to which they are attached;    -   R⁶ is selected from —H and -methyl;    -   each R⁸ is independently selected from —OH, -methoxy and        -ethoxy;    -   R¹⁰ is selected from

-   -   R²⁴ is selected from H and —C(O)R²⁵; wherein R²⁵ is selected        from —C₁-C₈ alkyl, —C₃-C₈ carbocycle, -aryl, —C₁-C₈ alkyl-aryl,        —C₁-C₈ alkyl-(C₃-C₈ carbocycle), —C₃-C₈ heterocycle and —C₁-C₈        alkyl-(C₃-C₈ heterocycle);    -   R²⁶ is selected from —C₁-C₈ alkyl, —C₃-C₈ carbocycle, -aryl,        —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈ carbocycle), —C₃-C₈        heterocycle and —C₁-C₈ alkyl-(C₃-C₈ heterocycle);    -   Z is —O—, —NH—, —OC(O)—, —NHC(O)—, —N(R²⁸)C(O)—; where R²⁸ is        selected from —H and —C₁-C₈ alkyl;    -   n is 0 or 1; and    -   R²⁷ is selected from —H, —N₃, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,        -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈ carbocycle),        —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈ heterocycle) when n        is 0; and R²⁷ is selected from —H, —C₁-C₈ alkyl, —C₃-C₈        carbocycle, -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈        carbocycle), —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈        heterocycle) when n is 1.

In one embodiment, R¹⁰ is selected from

In another embodiment, -D is represented by the formula:

-   -   wherein, independently at each location:    -   R¹ is selected from —H, —C₁-C₈ alkyl and —C₃-C₈ carbocycle; and        R² is selected from —H and —C₁-C₈ alkyl; or R¹ and R² join, have        the formula —(CR^(a)R^(b))_(n)— wherein R^(a) and R^(b) are        independently selected from —H, —C₁-C₈ alkyl and —C₃-C₈        carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a        ring with the nitrogen atom to which they are attached;    -   R³ is selected from —H, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,        —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈        carbocycle), —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈        heterocycle);    -   R⁴ is selected from —H, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,        —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈        carbocycle), —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈        heterocycle) wherein R⁵ is selected from —H and -methyl; or R⁴        and R⁵ join, have the formula —(CR^(a)R^(b))_(n)— wherein R^(a)        and R^(b) are independently selected from —H, —C₁-C₈ alkyl and        —C₃-C₈ carbocycle and n is selected from 2, 3, 4, 5 and 6, and        form a ring with the carbon atom to which they are attached;    -   R⁶ is selected from —H and —C₁-C₈ alkyl;    -   R⁷ is selected from —H, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,        —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈        carbocycle), —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈        heterocycle);    -   each R⁸ is independently selected from —H, —OH, —C₁-C₈ alkyl,        —C₃-C₈ carbocycle and —O—(C₁-C₈ alkyl);    -   R⁹ is selected from —H and —C₁-C₈ alkyl;    -   R¹⁰ is selected from

-   -   X is —O—, —S—, —NH— or —N(R¹⁴)—, where X forms a bond with a        Linker unit;    -   Z is —O—, —S—, —NH— or —N(R¹⁴)—;    -   R¹¹ is selected from —H, —OH, —NH₂, —NHR¹⁴, —N(R¹⁴)₂, —C₁-C₈        alkyl, —C₃-C₈ carbocycle, —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈        alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈ carbocycle), —C₃-C₈ heterocycle        and —C₁-C₈ alkyl-(C₃-C₈ heterocycle); or R¹¹ is an oxygen atom        which forms a carbonyl unit (C═O) with the carbon atom to which        it is attached and a hydrogen atom on this carbon atom is        replaced by one of the bonds in the (C═O) double bond;    -   each R¹² is independently selected from -aryl and —C₃-C₈        heterocycle;    -   R¹³ is selected from —H, —OH, —NH₂, —NHR¹⁴, —N(R¹⁴)₂, —O—(C₁-C₈        alkyl), —C₃-C₈ carbocycle, —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈        alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈ carbocycle), C₃-C₈ heterocycle        and —C₁-C₈ alkyl-(C₃-C₈ heterocycle);    -   each R¹⁴ is independently —H or —C₁-C₈ alkyl; and    -   R¹⁵ is -arylene-, —C₃-C₈ carbocyclo- or —C₃-C₈ heterocyclo-.

In one embodiment, when R¹ is —H, R¹⁰ is selected from:

In a preferred embodiment, -D has the formula

-   -   or a pharmaceutically acceptable salt or solvate thereof,    -   wherein, independently at each location:    -   R¹ is selected from —H and -methyl;    -   R² is selected from —H and -methyl;    -   R³ is selected from —H, -methyl, and -isopropyl;    -   R⁴ is selected from —H and -methyl; R⁵ is selected from        -isopropyl, -isobutyl, -sec-butyl, -methyl and -t-butyl; or R⁴        and R⁵ join, have the formula —(CR^(a)R^(b))_(n)— where R^(a)        and R^(b) are independently selected from —H, —C₁-C₈ alkyl, and        —C₃-C₈ carbocycle, and N is selected from 2, 3, 4, 5 and 6, and        form a ring with the carbon atom to which they are attached;    -   R⁶ is selected from —H and -methyl;    -   each R⁸ is independently selected from —OH, -methoxy and        -ethoxy;

-   R¹⁰ is selected from

where X is —O—, —NH— or —N(R¹⁴)— and forms a bond with Y when y is 1 or2, with W when y is 0, and with A when w and y are both 0;

Z is —O—, —NH— or —N(R¹⁴)—;

R¹³ is —H or -methyl;

R¹⁴ is C₁-C₈ alkyl; and

R¹⁵ is -arylene-, —C₃-C₈ carbocyclo or —C₃-C₈ heterocyclo-,

In one embodiment, when R¹ is -methyl, R¹⁰ is selected from

where X is —O—, —NH— or —N(R¹⁴)— and forms a bond with Y when y is 1 or2, and with W when y is 0;

Z is —O—, —NH— or —N(R¹⁴)—;

R¹³ is —H or -methyl;

R¹⁴ is C₁-C₈ alkyl; and

R¹⁵ is -arylene-, —C₃-C₈ carbocyclo or —C₃-C₈ heterocyclo-.

In another embodiment, when R¹ is —H, R¹⁰ is selected from:

where X is —O—, —NH— or —N(R¹⁴)— and forms a bond with Y when y is 1 or2, and with W when y is 0;

Z is —O—, —NH— or —N(R¹⁴)—;

R¹³ is —H or -methyl;

R¹⁴ is C₁-C₈ alkyl; and

R¹⁵ is -arylene-, —C₃-C₈ carbocyclo or —C₃-C₈ heterocyclo-.

A Drug unit can form a bond with a Linker unit via a nitrogen atom of aDrug's primary or secondary amino group, via an oxygen atom of a Drug'shydroxyl group, or via a sulfur atom of a Drug's sulfhydryl group toform a Drug-Linker Compound.

In a preferred embodiment, Drug units have the formula

The Ligand Unit

The Ligand unit (L-) includes within its scope any unit of a Ligand (L)that binds or reactively associates or complexes with a receptor,antigen or other receptive moiety associated with a given target-cellpopulation. A Ligand can be any molecule that binds to, complexes withor reacts with a moiety of a cell population sought to betherapeutically or otherwise biologically modified. The Ligand unit actsto deliver the Drug unit to the particular target cell population withwhich the Ligand unit reacts. Such Ligands include, but are not limitedto, large molecular weight proteins such as, for example, full-lengthantibodies, antibody fragments, smaller molecular weight proteins,polypeptide or peptides, and lectins.

A Ligand unit can form a bond to either a Stretcher unit or an AminoAcid unit of a Linker. A Ligand unit can form a bond to a Linker unitvia a heteroatom of the Ligand. Heteroatoms that may be present on aLigand unit include sulfur (in one embodiment, from a sulfhydryl groupof a Ligand), oxygen (in one embodiment, from a carbonyl, carboxyl orhydroxyl group of a Ligand) and nitrogen (in one embodiment, from aprimary or secondary amino group of a Ligand). These heteroatoms can bepresent on the Ligand in the Ligand's natural state, for example anaturally occurring antibody, or can be introduced into the Ligand viachemical modification.

In a preferred embodiment, a Ligand has a sulfhydryl group and theLigand bonds to the Linker unit via the sulfhydryl group's sulfur atom.

In another embodiment, the Ligand can have one or more carbohydrategroups that can be chemically modified to have one or more sulfhydrylgroups. The Ligand unit bonds to the Stretcher unit via the sulfhydrylgroup's sulfur atom.

In yet another embodiment, the Ligand can have one or more carbohydrategroups that can be oxidized to provide an aldehyde (—CHO) group (seeLaguzza, et al., J. Med. Chem. 1989, 32(3), 548-55). The correspondingaldehyde can form a bond with a Reactive Site on a Stretcher. Reactivesites on a Stretcher that can react with a carbonyl group on a Ligandinclude, but are not limited to, hydrazine and hydroxylamine.

Useful non-immunoreactive protein, polypeptide, or peptide Ligandsinclude, but are not limited to, transferrin, epidermal growth factors(“EGF”), bombesin, gastrin, gastrin-releasing peptide, platelet-derivedgrowth factor, IL-2, IL-6, transforming growth factors (“TGF”), such asTGF-α and TGF-β, vaccinia growth factor (“VGF”), insulin andinsulin-like growth factors I and II, lectins and apoprotein from lowdensity lipoprotein.

Useful Polyclonal antibody Ligands are heterogeneous populations ofantibody molecules derived from the sera of immunized animals. Variousprocedures well known in the art may be used for the production ofpolyclonal antibodies to an antigen-of-interest. For example, for theproduction of polyclonal antibodies, various host animals can beimmunized by injection with an antigen of interest or derivativethereof, including but not limited to rabbits, mice, rats, and guineapigs. Various adjuvants may be used to increase the immunologicalresponse, depending on the host species, and including but not limitedto Freund's (complete and incomplete), mineral gels such as aluminumhydroxide, surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanins, dinitrophenol, and potentially useful human adjuvants suchas BCG (bacille Calmette-Guerin) and corynebacterium parvum. Suchadjuvants are also well known in the art.

Useful monoclonal antibody Ligands are homogeneous populations ofantibodies to a particular antigen (e.g., a cancer cell antigen, a viralantigen, a microbial antigen covalently linked to a second molecule). Amonoclonal antibody (mAb) to an antigen-of-interest can be prepared byusing any technique known in the art which provides for the productionof antibody molecules by continuous cell lines in culture. Theseinclude, but are not limited to, the hybridoma technique originallydescribed by Kohler and Milstein (1975, Nature 256, 495-497), the humanB cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), and the EBV-hybridoma technique (Cole et al., 1985, MonoclonalAntibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Suchantibodies may be of any immunoglobulin class including IgG, IgM, IgE,IgA, and IgD and any subclass thereof. The hybridoma producing the mAbsof use in this invention may be cultivated in vitro or in vivo.

Useful monoclonal antibody Ligands include, but are not limited to,human monoclonal antibodies or chimeric human-mouse (or other species)monoclonal antibodies. Human monoclonal antibodies may be made by any ofnumerous techniques known in the art (e.g., Teng et al., 1983, Proc.Natl. Acad. Sci. U.S.A. 80, 7308-7312; Kozbor et al., 1983, ImmunologyToday 4, 72-79; and Olsson et al., 1982, Meth. Enzymol. 92, 3-16).

The Ligand can also be a bispecific antibody. Methods for makingbispecific antibodies are known in the art. Traditional production offull-length bispecific antibodies is based on the coexpression of twoimmunoglobulin heavy chain-light chain pairs, where the two chains havedifferent specificities (Milstein et al., 1983, Nature 305:537-539).Because of the random assortment of immunoglobulin heavy and lightchains, these hybridomas (quadromas) produce a potential mixture of 10different antibody molecules, of which only one has the correctbispecific structure. Purification of the correct molecule, which isusually performed using affinity chromatography steps, is rathercumbersome, and the product yields are low. Similar procedures aredisclosed in International Publication No. WO 93/08829, and inTraunecker et al., EMBO J. 10:3655-3659 (1991).

According to a different and more preferred approach, antibody variabledomains with the desired binding specificities (antibody-antigencombining sites) are fused to immunoglobulin constant domain sequences.The fusion preferably is with an immunoglobulin heavy chain constantdomain, comprising at least part of the hinge, CH_(2,) and CH3 regions.It is preferred to have the first heavy-chain constant region (CH1)containing the site necessary for light chain binding, present in atleast one of the fusions. DNAs encoding the immunoglobulin heavy chainfusions and, if desired, the immunoglobulin light chain, are insertedinto separate expression vectors, and are co-transfected into a suitablehost organism. This provides for great flexibility in adjusting themutual proportions of the three polypeptide fragments in embodimentswhen unequal ratios of the three polypeptide chains used in theconstruction provide the optimum yields. It is, however, possible toinsert the coding sequences for two or all three polypeptide chains inone expression vector when the expression of at least two polypeptidechains in equal ratios results in high yields or when the ratios are ofno particular significance.

In a preferred embodiment of this approach, the bispecific antibodieshave a hybrid immunoglobulin heavy chain with a first bindingspecificity in one arm, and a hybrid immunoglobulin heavy chain-lightchain pair (providing a second binding specificity) in the other arm.This asymmetric structure facilitates the separation of the desiredbispecific compound from unwanted immunoglobulin chain combinations, asthe presence of an immunoglobulin light chain in only one half of thebispecific molecule provides for a facile way of separation(International Publication No. WO 94/04690) which is incorporated hereinby reference in its entirety.

For further details for generating bispecific antibodies see, forexample, Suresh et al., Methods in Enzymology, 1986, 121:210. Using suchtechniques, bispecific antibody Ligands can be prepared for use in thetreatment or prevention of disease as defined herein.

Bifunctional antibodies are also described, in European PatentPublication No. EPA 0 105 360. As disclosed in this reference, hybrid orbifunctional antibodies can be derived either biologically, i.e., bycell fusion techniques, or chemically, especially with cross-linkingagents or disulfide-bridge forming reagents, and may comprise wholeantibodies or fragments thereof. Methods for obtaining such hybridantibodies are disclosed for example, in International Publication WO83/03679, and European Patent Publication No. EPA 0 217 577, both ofwhich are incorporated herein by reference.

The Ligand can be a functionally active fragment, derivative or analogof an antibody that immunospecifically binds to cancer cell antigens,viral antigens, or microbial antigens. In this regard, “Functionallyactive” means that the fragment, derivative or analog is able to elicitanti-anti-idiotype antibodies that recognize the same antigen that theantibody from which the fragment, derivative or analog is derivedrecognized. Specifically, in a preferred embodiment the antigenicity ofthe idiotype of the immunoglobulin molecule can be enhanced by deletionof framework and CDR sequences that are C-terminal to the CDR sequencethat specifically recognizes the antigen. To determine which CDRsequences bind the antigen, synthetic peptides containing the CDRsequences can be used in binding assays with the antigen by any bindingassay method known in the art (e.g., the BIA core assay)

Other useful Ligands include fragments of antibodies such as, but notlimited to, F(ab′)2 fragments, which contain the variable region, thelight chain constant region and the CH1 domain of the heavy chain can beproduced by pepsin digestion of the antibody molecule, and Fabfragments, which can be generated by reducing the disulfide bridges ofthe F(ab′)2 fragments. Other useful Ligands are heavy chain and lightchain dimers of antibodies, or any minimal fragment thereof such as Fvsor single chain antibodies (SCAs) (e.g., as described in U.S. Pat. No.4,946,778; Bird, 1988, Science 242:423-42; Huston et al., 1988, Proc.Natl. Acad. Sci. USA 85:5879-5883; and Ward et al., 1989, Nature334:544-54), or any other molecule with the same specificity as theantibody.

Additionally, recombinant antibodies, such as chimeric and humanizedmonoclonal antibodies, comprising both human and non-human portions,which can be made using standard recombinant DNA techniques, are usefulLigands. A chimeric antibody is a molecule in which different portionsare derived from different animal species, such as those having avariable region derived from a murine monoclonal and a humanimmunoglobulin constant region. (See, e.g., Cabilly et al., U.S. Pat.No. 4,816,567; and Boss et al., U.S. Pat. No. 4,816,397, which areincorporated herein by reference in their entirety.) Humanizedantibodies are antibody molecules from non-human species having one ormore complementarity determining regions (CDRs) from the non-humanspecies and a framework region from a human immunoglobulin molecule.(See, e.g., Queen, U.S. Pat. No. 5,585,089, which is incorporated hereinby reference in its entirety.) Such chimeric and humanized monoclonalantibodies can be produced by recombinant DNA techniques known in theart, for example using methods described in International PublicationNo. WO 87/02671; European Patent Publication No. 184,187; EuropeanPatent Publication No. 171,496; European Patent Publication No. 173,494;International Publication No. WO 86/01533; U.S. Pat. No. 4,816,567;European Patent Publication No. 125,023; Berter et al., 1988, Science240:1041-1043; Liu et al., 1987, Proc. Natl. Acad. Sci. USA84:3439-3443; Liu et al., 1987, J. Immunol. 139:3521-3526; Sun et al.,1987, Proc. Natl. Acad. Sci. USA 84:214-218; Nishimura et al., 1987,Canc. Res. 47:999-1005; Wood et al., 1985, Nature 314:446-449; and Shawet al., 1988, J. Natl. Cancer Inst. 80:1553-1559; Morrison, 1985,Science 229:1202-1207; Oi et al., 1986, BioTechniques 4:214; U.S. Pat.No. 5,225,539; Jones et al., 1986, Nature 321:552-525; Verhoeyan et al.(1988) Science 239:1534; and Beidler et al., 1988, J. Immunol.141:4053-4060; each of which is incorporated herein by reference in itsentirety.

Completely human antibodies are particularly desirable for Ligands. Suchantibodies can be produced using transgenic mice that are incapable ofexpressing endogenous immunoglobulin heavy and light chains genes, butwhich can express human heavy and light chain genes. The transgenic miceare immunized in the normal fashion with a selected antigen, e.g., allor a portion of a polypeptide of the invention. Monoclonal antibodiesdirected against the antigen can be obtained using conventionalhybridoma technology. The human immunoglobulin transgenes harbored bythe transgenic mice rearrange during B cell differentiation, andsubsequently undergo class switching and somatic mutation. Thus, usingsuch a technique, it is possible to produce therapeutically useful IgG,IgA, IgM and IgE antibodies. For an overview of this technology forproducing human antibodies, see Lonberg and Huszar (1995, Int. Rev.Immunol. 13:65-93). For a detailed discussion of this technology forproducing human antibodies and human monoclonal antibodies and protocolsfor producing such antibodies, see, e.g., U.S. Pat. No. 5,625,126; U.S.Pat. No. 5,633,425; U.S. Pat. No. 5,569,825; U.S. Pat. No. 5,661,016;and U.S. Pat. No. 5,545,806; each of which is incorporated herein byreference in its entirety. Other human antibodies can be obtainedcommercially from, for example, Abgenix, Inc. (Freemont, Calif.) andGenpharm (San Jose, Calif.).

Completely human antibodies that recognize a selected epitope can begenerated using a technique referred to as “guided selection.” In thisapproach a selected non-human monoclonal antibody, e.g., a mouseantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope. (Jespers et al. (1994) Biotechnology12:899-903).

In other embodiments, the Ligand is a fusion protein of an antibody, ora functionally active fragment thereof, for example in which theantibody is fused via a covalent bond (e.g., a peptide bond), at eitherthe N-terminus or the C-terminus to an amino acid sequence of anotherprotein (or portion thereof, preferably at least 10, 20 or 50 amino acidportion of the protein) that is not the antibody. Preferably, theantibody or fragment thereof is covalently linked to the other proteinat the N-terminus of the constant domain.

The Ligand antibodies include analogs and derivatives that are eithermodified, i.e, by the covalent attachment of any type of molecule aslong as such covalent attachment permits the antibody to retain itsantigen binding immunospecificity. For example, but not by way oflimitation, the derivatives and analogs of the antibodies include thosethat have been further modified, e.g., by glycosylation, acetylation,pegylation, phosphylation, amidation, derivatization by knownprotecting/blocking groups, proteolytic cleavage, linkage to a cellularLigand unit or other protein, etc. Any of numerous chemicalmodifications can be carried out by known techniques, including, but notlimited to specific chemical cleavage, acetylation, formylation,metabolic synthesis of tunicamycin, etc. Additionally, the analog orderivative can contain one or more unnaturalamino acids.

The Ligand antibodies include antibodies having modifications (e.g.,substitutions, deletions or additions) in amino acid residues thatinteract with Fc receptors. In particular, the Ligand antibodies includeantibodies having modifications in amino acid residues identified asinvolved in the interaction between the Fc domain and the FcRn receptor(see, e.g., International Publication No. WO 97/34631, which isincorporated herein by reference in its entirety). Antibodiesimmunospecific for a cancer cell antigen can be obtained commercially,for example, from Genentech (San Francisco, Calif.) or produced by anymethod known to one of skill in the art such as, e.g., chemicalsynthesis or recombinant expression techniques. The nucleotide sequenceencoding antibodies immunospecific for a cancer cell antigen can beobtained, e.g., from the GenBank database or a database like it, theliterature publications, or by routine cloning and sequencing.

In a specific embodiment, known antibodies for the treatment orprevention of cancer are used in accordance with the compositions andmethods of the invention. Antibodies immunospecific for a cancer cellantigen can be obtained commercially or produced by any method known toone of skill in the art such as, e.g., chemical synthesis or recombinantexpression techniques. The nucleotide sequence encoding antibodiesimmunospecific for a cancer cell antigen can be obtained, e.g., from theGenBank database or a database like it, the literature publications, orby routine cloning and sequencing. Examples of antibodies available forthe treatment of cancer include, but are not limited to, HERCEPTIN(Trastuzumab; Genentech, CA) which is a humanized anti-HER2 monoclonalantibody for the treatment of patients with metastatic breast cancer(Stebbing, J., Copson, E., and O'Reilly, S. “Herceptin (trastuzamab) inadvanced breast cancer” Cancer Treat Rev. 26, 287-90, 2000); RITUXAN(rituximab; Genentech) which is a chimeric anti-CD20 monoclonal antibodyfor the treatment of patients with non-Hodgkin's lymphoma; OvaRex(AltaRex Corporation, MA) which is a murine antibody for the treatmentof ovarian cancer; Panorex (Glaxo Wellcome, NC) which is a murineIgG_(2a) antibody for the treatment of colorectal cancer; BEC2 (ImCloneSystems Inc., NY) which is murine IgG antibody for the treatment of lungcancer; IMC-C225 (Imclone Systems Inc., NY) which is a chimeric IgGantibody for the treatment of head and neck cancer; Vitaxin (MedImmune,Inc., MD) which is a humanized antibody for the treatment of sarcoma;Campath I/H (Leukosite, MA) which is a humanized IgG₁ antibody for thetreatment of chronic lymphocytic leukemia (CLL); Smart MI95 (ProteinDesign Labs, Inc., CA) which is a humanized IgG antibody for thetreatment of acute myeloid leukemia (AML); LymphoCide (Immunomedics,Inc., NJ) which is a humanized IgG antibody for the treatment ofnon-Hodgkin's lymphoma; Smart ID10 (Protein Design Labs, Inc., CA) whichis a humanized antibody for the treatment of non-Hodgkin's lymphoma;Oncolym (Techniclone, Inc., CA) which is a murine antibody for thetreatment of non-Hodgkin's lymphoma; Allomune (BioTransplant, CA) whichis a humanized anti-CD2 mAb for the treatment of Hodgkin's Disease ornon-Hodgkin's lymphoma; anti-VEGF (Genentech, Inc., CA) which ishumanized antibody for the treatment of lung and colorectal cancers;CEAcide (Immunomedics, NJ) which is a humanized anti-CEA antibody forthe treatment of colorectal cancer; IMC-1C11 (ImClone Systems, NJ) whichis an anti-KDR chimeric antibody for the treatment of colorectal cancer,lung cancers, and melanoma; and Cetuximab (ImClone, NJ) which is ananti-EGFR chimeric antibody for the treatment of epidermal growth factorpositive cancers.

Other antibodies useful in the treatment of cancer include, but are notlimited to, antibodies against the following antigens: CA125 (ovarian),CA15-3 (carcinomas), CA19-9 (carcinomas), L6 (carcinomas), Lewis Y(carcinomas), Lewis X (carcinomas), alpha fetoprotein (carcinomas), CA242 (colorectal), placental alkaline phosphatase (carcinomas), prostatespecific antigen (prostate), prostatic acid phosphatase (prostate),epidermal growth factor (carcinomas), MAGE-1 (carcinomas), MAGE-2(carcinomas), MAGE-3 (carcinomas), MAGE-4 (carcinomas), anti-transferrinreceptor (carcinomas), p97 (melanoma), MUC1-KLH (breast cancer), CEA(colorectal), gp100 (melanoma), MART1 (melanoma), PSA (prostate), IL-2receptor (T-cell leukemia and lymphomas), CD20 (non-Hodgkin's lymphoma),CD52 (leukemia), CD33 (leukemia), CD22 (lymphoma), human chorionicgonadotropin (carcinoma), CD38 (multiple myeloma), CD40 (lymphoma),mucin (carcinomas), P21 (carcinomas), MPG (melanoma), and Neu oncogeneproduct (carcinomas). Some specific useful antibodies include, but arenot limited to, BR96 mAb (Trail, P. A., Willner, D., Lasch, S. J.,Henderson, A. J., Hofstead, S. J., Casazza, A. M., Firestone, R. A.,Hellström, I., Hellström, K. E., “Cure of Xenografted Human Carcinomasby BR96-Doxorubicin Immunoconjugates” Science 1993, 261, 212-215), BR64(Trail, P A, Willner, D, Knipe, J., Henderson, A. J., Lasch, S. J.,Zoeckler, M. E., Trailsmith, M. D., Doyle, T. W., King, H. D., Casazza,A. M., Braslawsky, G. R., Brown, J. P., Hofstead, S. J., (Greenfield, R.S., Firestone, R. A., Mosure, K., Kadow, D. F., Yang, M. B., Hellstrom,K. E., and Hellstrom, I. “Effect of Linker Variation on the Stability,Potency, and Efficacy of Carcinoma-reactive BR64-DoxorubicinImmunoconjugates” Cancer Research 1997, 57, 100-105, mAbs against theCD40 antigen, such as S2C6 mAb (Francisco, J. A., Donaldson, K. L.,Chace, D., Siegall, C. B., and Wahl, A. F. “Agonistic properties and invivo antitumor activity of the anti-CD-40 antibody, SGN-14” Cancer Res.2000, 60, 3225-3231), mAbs against the CD70 antigen, such as 1F6 mAb,and mAbs against the CD30 antigen, such as AC10 (Bowen, M. A., Olsen, K.J., Cheng, L., Avila, D., and Podack, E. R. “Functional effects of CD30on a large granular lymphoma cell line YT” J. Immunol., 151, 5896-5906,1993). Many other internalizing antibodies that bind to tumor associatedantigens can be used in this invention, and have been reviewed (Franke,A. E., Sievers, E. L., and Scheinberg, D. A., “Cell surfacereceptor-targeted therapy of acute myeloid leukemia: a review” CancerBiother Radiopharm. 2000, 15, 459-76; Murray, J. L., “Monoclonalantibody treatment of solid tumors: a coming of age” Semin Oncol. 2000,27, 64-70; Breitling, F., and Dubel, S., Recombinant Antibodies, JohnWiley, and Sons, New York, 1998).

In another specific embodiment, known antibodies for the treatment orprevention of an autoimmune disease are used in accordance with thecompositions and methods of the invention. Antibodies immunospecific foran antigen of a cell that is responsible for producing autoimmuneantibodies can be obtained from any organization (e.g., a universityscientist or a company such as Genentech) or produced by any methodknown to one of skill in the art such as, e.g., chemical synthesis orrecombinant expression techniques. In another embodiment, useful Ligandantibodies that are immunospecific for the treatment of autoimmunediseases include, but are not limited to, Anti-Nuclear Antibody; Anti dsDNA; Anti ss DNA, Anti Cardiolipin Antibody IgM, IgG; Anti PhospholipidAntibody IgM, IgG; Anti SM Antibody; Anti Mitochondrial Antibody;Thyroid Antibody; Microsomal Antibody; Thyroglobulin Antibody; AntiSCL-70; Anti-Jo; Anti-U₁RNP; Anti-La/SSB; Anti SSA; Anti SSB; AntiPerital Cells Antibody; Anti Histones; Anti RNP; C-ANCA; P-ANCA; Anticentromere; Anti-Fibrillarin, and Anti GBM Antibody.

In certain preferred embodiments, antibodies useful in the presentmethods, can bind to both a receptor or a receptor complex expressed onan activated lymphocyte. The receptor or receptor complex can comprisean immunoglobulin gene superfamily member, a TNF receptor superfamilymember, an integrin, a cytokine receptor, a chemokine receptor, a majorhistocompatibility protein, a lectin, or a complement control protein.Non-limiting examples of suitable immunoglobulin superfamily members areCD2, CD3, CD4, CD8, CD19, CD22, CD28, CD79, CD90, CD152/CTLA-4, PD-1,and ICOS. Non-limiting examples of suitable TNF receptor superfamilymembers are CD27, CD40, CD95/Fas, CD134/OX40, CD137/4-1BB, TNF-R1,TNFR-2, RANK, TACI, BCMA, osteoprotegerin, Apo2/TRAIL-R1, TRAIL-R2,TRAIL-R3, TRAIL-R4, and APO-3. Non-limiting examples of suitableintegrins are CD11a, CD11b, CD11c, CD18, CD29, CD41, CD49a, CD49b,CD49c, CD49d, CD49e, CD49f, CD103, and CD104. Non-limiting examples ofsuitable lectins are C-type, S-type, and I-type lectin.

In one embodiment, the Ligand is an antibody that binds to an activatedlymphocyte that is associated with an autoimmune disease.

In another specific embodiment, useful Ligand antibodies that areimmunospecific for a viral or a microbial antigen are monoclonalantibodies. Preferably, Ligand antibodies that are immunospecific for aviral antigen or microbial antigen are humanized or human monoclonalantibodies. As used herein, the term “viral antigen” includes, but isnot limited to, any viral peptide, polypeptide protein (e.g., HIV gp120,HIV nef, RSV F glycoprotein, influenza virus neuraminidase, influenzavirus hemagglutinin, HTLV tax, herpes simplex virus glycoprotein (e.g.,gB, gC, gD, and gE) and hepatitis B surface antigen) that is capable ofeliciting an immune response. As used herein, the term “microbialantigen” includes, but is not limited to, any microbial peptide,polypeptide, protein, saccharide, polysaccharide, or lipid molecule(e.g., a bacterial, fungi, pathogenic protozoa, or yeast polypeptideincluding, e.g., LPS and capsular polysaccharide 5/8) that is capable ofeliciting an immune response.

Antibodies immunospecific for a viral or microbial antigen can beobtained commercially, for example, from Genentech (San Francisco,Calif.) or produced by any method known to one of skill in the art suchas, e.g., chemical synthesis or recombinant expression techniques. Thenucleotide sequence encoding antibodies that are immunospecific for aviral or microbial antigen can be obtained, e.g., from the GenBankdatabase or a database like it, the literature publications, or byroutine cloning and sequencing.

In a specific embodiment, useful Ligand antibodies are those that areuseful for the treatment or prevention of viral or microbial infectionin accordance with the methods of the invention. Examples of antibodiesavailable useful for the treatment of viral infection or microbialinfection include, but are not limited to, SYNAGIS (MedImmune, Inc., MD)which is a humanized anti-respiratory syncytial virus (RSV) monoclonalantibody useful for the treatment of patients with RSV infection; PRO542(Progenics) which is a CD4 fusion antibody useful for the treatment ofHIV infection; OSTAVIR (Protein Design Labs, Inc., CA) which is a humanantibody useful for the treatment of hepatitis B virus; PROTOVIR(Protein Design Labs, Inc., CA) which is a humanized IgG₁ antibodyuseful for the treatment of cytomegalovirus (CMV); and anti-LPSantibodies.

Other antibodies useful in the treatment of infectious diseases include,but are not limited to, antibodies against the antigens from pathogenicstrains of bacteria (Streptococcus pyogenes, Streptococcus pneumoniae,Neisseria gonorrheae, Neisseria meningitidis, Corynebacteriumdiphtheriae, Clostridium botulinum, Clostridium perfringens, Clostridiumtetani, Hemophilus influenzae, Klebsiella pneumoniae, Klebsiellaozaenas, Klebsiella rhinoscleromotis, Staphylococcus aureus, Vibriocolerae, Escherichia coli, Pseudomonas aeruginosa, Campylobacter(Vibrio) fetus, Aeromonas hydrophila, Bacillus cereus, Edwardsiellatarda, Yersinia enterocolitica, Yersinia pestis, Yersiniapseudotuberculosis, Shigella dysenteriae, Shigella flexneri, Shigellasonnei, Salmonella typhimurium, Treponema pallidum, Treponema pertenue,Treponema carateneum, Borrelia vincentii, Borrelia burgdorferi,Leptospira icterohemorrhagiae, Mycobacterium tuberculosis, Pneumocystiscarinii, Francisella tularensis, Brucella abortus, Brucella suis,Brucella melitensis, Mycoplasma spp., Rickettsia prowazeki, Rickettsiatsutsugumushi, Chlamydia spp.); pathogenic fungi (Coccidioides immitis,Aspergillus fumigatus, Candida albicans, Blastomyces dermatitidis,Cryptococcus neoformans, Histoplasma capsulatum); protozoa (Entomoebahistolytica, Toxoplasma gondii, Trichomonas tenas, Trichomonas hominis,Trichomonas vaginalis, Tryoanosoma gambiense, Trypanosoma rhodesiense,Trypanosoma cruzi, Leishmania donovani, Leishmania tropica, Leishmaniabraziliensis, Pneumocystis pneumonia, Plasmodium vivax, Plasmodiumfalciparum, Plasmodium malaria); or Helminiths (Enterobius vermicularis,Trichuris trichiura, Ascaris lumbricoides, Trichinella spiralis,Strongyloides stercoralis, Schistosoma japonicum, Schistosoma mansoni,Schistosoma haematobium, and hookworms).

Other antibodies useful in this invention for treatment of viral diseaseinclude, but are not limited to, antibodies against antigens ofpathogenic viruses, including as examples and not by limitation:Poxviridae, Herpesviridae, Herpes Simplex virus 1, Herpes Simplex virus2, Adenoviridae, Papovaviridae, Enteroviridae, Picornaviridae,Parvoviridae, Reoviridae, Retroviridae, influenza viruses, parainfluenzaviruses, mumps, measles, respiratory syncytial virus, rubella,Arboviridae, Rhabdoviridae, Arenaviridae, Hepatitis A virus, Hepatitis Bvirus, Hepatitis C virus, Hepatitis E virus, Non-A/Non-B Hepatitisvirus, Rhinoviridae, Coronaviridae, Rotoviridae, and HumanImmunodeficiency Virus.

The antibodies suitable for use in the invention can be produced by anymethod known in the art for the synthesis of antibodies, in particular,by chemical synthesis or by recombinant expression, and are preferablyproduced by recombinant expression techniques.

Production of Recombinant Antibodies

Ligand antibodies of the invention can be produced using any methodknown in the art to be useful for the synthesis of antibodies, inparticular, by chemical synthesis or by recombinant expression, and arepreferably produced by recombinant expression techniques.

Recombinant expression of the Ligand antibodies, or fragment, derivativeor analog thereof, requires construction of a nucleic acid that encodesthe antibody. If the nucleotide sequence of the antibody is known, anucleic acid encoding the antibody may be assembled from chemicallysynthesized oligonucleotides (e.g., as described in Kutmeier et al.,1994, BioTechniques 17:242), which involves the synthesis of overlappingoligonucleotides containing portions of the sequence encoding theantibody, annealing and ligation of those oligonucleotides, and thenamplification of the ligated oligonucleotides by PCR.

Alternatively, a nucleic acid molecule encoding an antibody can begenerated from a suitable source. If a clone containing the nucleic acidencoding the particular antibody is not available, but the sequence ofthe antibody is known, a nucleic acid encoding the antibody can beobtained from a suitable source (e.g., an antibody cDNA library, or cDNAlibrary generated from any tissue or cells expressing theimmunoglobulin) by PCR amplification using synthetic primershybridizable to the 3′ and 5′ ends of the sequence or by cloning usingan oligonucleotide probe specific for the particular gene sequence.

If an antibody that specifically recognizes a particular antigen is notcommercially available (or a source for a cDNA library for cloning anucleic acid encoding such an immunoglobulin), antibodies specific for aparticular antigen can be generated by any method known in the art, forexample, by immunizing an animal, such as a rabbit, to generatepolyclonal antibodies or, more preferably, by generating monoclonalantibodies, e.g., as described by Kohler and Milstein (1975, Nature256:495-497) or, as described by Kozbor et al. (1983, Immunology Today4:72) or Cole et al. (1985 in Monoclonal Antibodies and Cancer Therapy,Alan R. Liss, Inc., pp. 77-96). Alternatively, a clone encoding at leastthe Fab portion of the antibody can be obtained by screening Fabexpression libraries (e.g., as described in Huse et al., 1989, Science246:1275-1281) for clones of Fab fragments that bind the specificantigen or by screening antibody libraries (See, e.g., Clackson et al.,1991, Nature 352:624; Hane et al., 1997 Proc. Natl. Acad. Sci. USA94:4937).

Once a nucleic acid sequence encoding at least the variable domain ofthe antibody is obtained, it can be introduced into a vector containingthe nucleotide sequence encoding the constant regions of the antibody(see, e.g., International Publication No. WO 86/05807; InternationalPublication No. WO 89/01036; and U.S. Pat. No. 5,122,464). Vectorscontaining the complete light or heavy chain that allow for theexpression of a complete antibody molecule are available. Then, thenucleic acid encoding the antibody can be used to introduce thenucleotide substitutions or deletion necessary to substitute (or delete)the one or more variable region cysteine residues participating in anintrachain disulfide bond with an amino acid residue that does notcontain a sulfhydyl group. Such modifications can be carried out by anymethod known in the art for the introduction of specific mutations ordeletions in a nucleotide sequence, for example, but not limited to,chemical mutagenesis and in vitro site directed mutagenesis (Hutchinsonet al., 1978, J. Biol. Chem. 253:6551).

In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci. 81:851-855;Neuberger et al., 1984, Nature 312:604-608; Takeda et al., 1985, Nature314:452-454) by splicing genes from a mouse antibody molecule ofappropriate antigen specificity together with genes from a humanantibody molecule of appropriate biological activity can be used. Achimeric antibody is a molecule in which different portions are derivedfrom different animal species, such as those having a variable regionderived from a murine monoclonal antibody and a human immunoglobulinconstant region, e.g., humanized antibodies.

Alternatively, techniques described for the production of single chainantibodies (U.S. Pat. No. 4,694,778; Bird, 1988, Science 242:423-42;Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Wardet al., 1989, Nature 334:544-54) can be adapted to produce single chainantibodies. Single chain antibodies are formed by linking the heavy andlight chain fragments of the Fv region via an amino acid bridge,resulting in a single chain polypeptide. Techniques for the assembly offunctional Fv fragments in E. coli may also be used (Skerra et al.,1988, Science 242:1038-1041).

Antibody fragments that recognize specific epitopes can be generated byknown techniques. For example, such fragments include, but are notlimited to, the F(ab′)2 fragments that can be produced by pepsindigestion of the antibody molecule and the Fab fragments that can begenerated by reducing the disulfide bridges of the F(ab′)2 fragments.

Once a nucleic acid sequence encoding a Ligand antibody has beenobtained, the vector for the production of the antibody can be producedby recombinant DNA technology using techniques well known in the art.Methods that are well known to those skilled in the art can be used toconstruct expression vectors containing the antibody coding sequencesand appropriate transcriptional and translational control signals. Thesemethods include, for example, in vitro recombinant DNA techniques,synthetic techniques, and in vivo genetic recombination. See, forexample, the techniques described in Sambrook et al. (1990, MolecularCloning, A Laboratory Manual, 2^(nd) Ed., Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.) and Ausubel et al. (eds., 1998, CurrentProtocols in Molecular Biology, John Wiley & Sons, NY).

An expression vector comprising the nucleotide sequence of an antibodyor the nucleotide sequence of an antibody can be transferred to a hostcell by conventional techniques (e.g., electroporation, liposomaltransfection, and calcium phosphate precipitation), and the transfectedcells are then cultured by conventional techniques to produce theantibody. In specific embodiments, the expression of the antibody isregulated by a constitutive, an inducible or a tissue, specificpromoter.

The host cells used to express the recombinant Ligand antibody can beeither bacterial cells such as Escherichia coli, or, preferably,eukaryotic cells, especially for the expression of whole recombinantimmunoglobulin molecule. In particular, mammalian cells such as Chinesehamster ovary cells (CHO), in conjunction with a vector such as themajor intermediate early gene promoter element from humancytomegalovirus is an effective expression system for immunoglobulins(Foecking et al., 198, Gene 45:101; Cockett et al., 1990, BioTechnology8:2).

A variety of host-expression vector systems can be utilized to expressthe immunoglobulin Ligands. Such host-expression systems representvehicles by which the coding sequences of the antibody can be producedand subsequently purified, but also represent cells that can, whentransformed or transfected with the appropriate nucleotide codingsequences, express a Ligand immunoglobulin molecule in situ. Theseinclude, but are not limited to, microorganisms such as bacteria (e.g.,E. coli and B. subtilis) transformed with recombinant bacteriophage DNA,plasmid DNA or cosmid DNA expression vectors containing immunoglobulincoding sequences; yeast (e.g., Saccharomyces Pichia) transformed withrecombinant yeast expression vectors containing immunoglobulin codingsequences; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing the immunoglobulincoding sequences; plant cell systems infected with recombinant virusexpression vectors (e.g., cauliflower mosaic virus (CaMV) and tobaccomosaic virus (TMV)) or transformed with recombinant plasmid expressionvectors (e.g., Ti plasmid) containing immunoglobulin coding sequences;or mammalian cell systems (e.g., COS, CHO, BH, 293, 293T, 3T3 cells)harboring recombinant expression constructs containing promoters derivedfrom the genome of mammalian cells (e.g., metallothionein promoter) orfrom mammalian viruses (e.g., the adenovirus late promoter; the vacciniavirus 7.5K promoter).

In bacterial systems, a number of expression vectors can beadvantageously selected depending upon the use intended for the antibodybeing expressed. For example, when a large quantity of such a protein isto be produced, vectors that direct the expression of high levels offusion protein products that are readily purified might be desirable.Such vectors include, but are not limited, to the E. coli expressionvector pUR278 (Ruther et al., 1983, EMBO J. 2:1791), in which theantibody coding sequence may be ligated individually into the vector inframe with the lac Z coding region so that a fusion protein is produced;pIN vectors (Inouye & Inouye, 1985, Nucleic Acids Res. 13:3101-3109; VanHeeke & Schuster, 1989, J. Biol. Chem. 24:5503-5509); and the like. pGEXvectors can also be used to express foreign polypeptides as fusionproteins with glutathione S-transferase (GST). In general, such fusionproteins are soluble and can easily be purified from lysed cells byadsorption and binding to a matrix glutathione-agarose beads followed byelution in the presence of free glutathione. The pGEX vectors aredesigned to include thrombin or factor Xa protease cleavage sites sothat the cloned target gene product can be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) or the analogous virus from Drosophila Melanogaster is used as avector to express foreign genes. The virus grows in Spodopterafrugiperda cells. The antibody coding sequence can be clonedindividually into non-essential regions (for example the polyhedringene) of the virus and placed under control of an AcNPV promoter (forexample the polyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems canbe utilized. In cases where an adenovirus is used as an expressionvector, the antibody coding sequence of interest can be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric gene can then beinserted in the adenovirus genome by in vitro or in vivo recombination.Insertion in a non-essential region of the viral genome (e.g., region E1or E3) results in a recombinant virus that is viable and capable ofexpressing the immunoglobulin molecule in infected hosts. (e.g., seeLogan & Shenk, 1984, Proc. Natl. Acad. Sci. USA 81:355-359). Specificinitiation signals can also be required for efficient translation ofinserted antibody coding sequences. These signals include the ATGinitiation codon and adjacent sequences. Furthermore, the initiationcodon must be in phase with the reading frame of the desired codingsequence to ensure translation of the entire insert. These exogenoustranslational control signals and initiation codons can be of a varietyof origins, both natural and synthetic. The efficiency of expression canbe enhanced by the inclusion of appropriate transcription enhancerelements, transcription terminators, etc. (see Bittner et al., 1987,Methods in Enzymol. 153:51-544).

In addition, a host cell strain can be chosen to modulate the expressionof the inserted sequences, or modifies and processes the gene product inthe specific fashion desired. Such modifications (e.g., glycosylation)and processing (e.g., cleavage) of protein products can be important forthe function of the protein. Different host cells have characteristicand specific mechanisms for the post-translational processing andmodification of proteins and gene products. Appropriate cell lines orhost systems can be chosen to ensure the correct modification andprocessing of the foreign protein expressed. To this end, eukaryotichost cells that possess the cellular machinery for proper processing ofthe primary transcript, glycosylation, and phosphorylation of the geneproduct can be used. Such mammalian host cells include, but are notlimited to, CHO, VERY, BH, Hela, COS, MDCK, 293, 293T, 3T3, W138, BT483,Hs578T, HTB2, BT20 and T47D, CRL7030 and Hs578Bst.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines that stably express anantibody can be engineered. Rather than using expression vectors thatcontain viral origins of replication, host cells can be transformed withDNA controlled by appropriate expression control elements (e.g.,promoter, enhancer, sequences, transcription terminators,polyadenylation sites, etc.), and a selectable marker. Following theintroduction of the foreign DNA, engineered cells can be allowed to growfor 1-2 days in an enriched media, and then are switched to a selectivemedia. The selectable marker in the recombinant plasmid confersresistance to the selection and allows cells to stably integrate theplasmid into their chromosomes and grow to form foci that in turn can becloned and expanded into cell lines. This method can advantageously beused to engineer cell lines which express the antibody Such engineeredcell lines can be particularly useful in screening and evaluation oftumor antigens that interact directly or indirectly with the antibodyLigand.

A number of selection systems can be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska &Szybalski, 192, Proc. Natl. Acad. Sci. USA 48:202), and adeninephosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genes can beemployed in tk-, hgprt- or aprt-cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., 1980, Proc. Natl. Acad. Sci. USA 77:357; O'Hare et al., 1981,Proc. Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA78:2072); neo, which confers resistance to the aminoglycoside G-418(Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy 3:87-95;Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan,1993, Science 260:926-932; and Morgan and Anderson, 1993, Ann. Rev.Biochem. 62:191-217; May, 1993, TIB TECH 11(5):155-215) and hygro, whichconfers resistance to hygromycin (Santerre et al., 1984, Gene 30:147).Methods commonly known in the art of recombinant DNA technology whichcan be used are described in Ausubel et al. (eds., 1993, CurrentProtocols in Molecular Biology, John Wiley & Sons, NY; Kriegler, 1990,Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY;and in Chapters 12 and 13, Dracopoli et al. (eds), 1994, CurrentProtocols in Human Genetics, John Wiley & Sons, NY.; Colberre-Garapin etal., 1981, J. Mol. Biol. 150:1).

The expression levels of an antibody can be increased by vectoramplification (for a review, see Bebbington and Hentschel, The use ofvectors based on gene amplification for the expression of cloned genesin mammalian cells in DNA cloning, Vol. 3. (Academic Press, New York,1987)). When a marker in the vector system expressing an antibody isamplifiable, an increase in the level of inhibitor present in culture ofhost cell will increase the number of copies of the marker gene. Sincethe amplified region is associated with the nucleotide sequence of theantibody, production of the antibody will also increase (Crouse et al.,1983, Mol. Cell. Biol. 3:257).

The host cell can be co-transfected with two expression vectors of theinvention, the first vector encoding a heavy chain derived polypeptideand the second vector encoding a light chain derived polypeptide. Thetwo vectors can contain identical selectable markers that enable equalexpression of heavy and light chain polypeptides. Alternatively, asingle vector can be used to encode both heavy and light chainpolypeptides. In such situations, the light chain should be placedbefore the heavy chain to avoid an excess of toxic free heavy chain(Proudfoot, 1986, Nature 322:52; Kohler, 1980, Proc. Natl. Acad. Sci.USA 77:2197). The coding sequences for the heavy and light chains cancomprise cDNA or genomic DNA.

Once the antibody has been recombinantly expressed, it can be purifiedusing any method known in the art for purification of an antibody, forexample, by chromatography (e.g., ion exchange, affinity, particularlyby affinity for the specific antigen after Protein A, and sizing columnchromatography), centrifugation, differential solubility, or by anyother standard technique for the purification of proteins.

In a preferred embodiment, the Ligand is an antibody.

In a more preferred embodiment, the Ligand is a monoclonal antibody.

In any case, the hybrid antibodies have a dual specificity, preferablywith one or more binding sites specific for the hapten of choice or oneor more binding sites specific for a target antigen, for example, anantigen associated with a tumor, an autoimmune disease, an infectiousorganism, or other disease state.

Synthesis of the Compounds of the Invention

As described in more detail below, the Compounds of the Invention areconveniently prepared using a Linker having two or more Reactive Sitesfor binding to the Drug and Ligand. In one aspect of the invention, aLinker has a Reactive site which has an electrophilic group that isreactive to a nucleophilic group present on a Ligand. Usefulnucleophilic groups on a Ligand include but are not limited to,sulfhydryl, hydroxyl and amino groups. The heteroatom of thenucleophilic group of a Ligand is reactive to an electrophilic group ona Linker and forms a covalent bond to a Linker unit. Usefulelectrophilic groups include, but are not limited to, maleimide andhaloacetamide groups. The electrophilic group provides a convenient sitefor Ligand attachment.

In another embodiment, a Linker has a Reactive site which has anucleophilic group that is reactive to an electrophilic group present ona Ligand. Useful electrophilic groups on a Ligand include, but are notlimited to, aldehyde and ketone carbonyl groups. The heteroatom of anucleophilic group of a Linker can react with an electrophilic group ona Ligand and form a covalent bond to a Ligand unit. Useful nucleophilicgroups on a Linker include, but are not limited to, hydrazide, oxime,amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, andarylhydrazide. The electrophilic group on a Ligand provides a convenientsite for attachment to a Linker.

Carboxylic acid functional groups and chloroformate functional groupsare also useful reactive sites for a Linker because they can react withprimary or secondary amino groups of a Drug to form an amide linkage.Also useful as a reactive site is a carbonate functional group on aLinker which can react with an amino group or hydroxyl group of a Drugto form a carbamate linkage or carbonate linkage, respectively.Similarly, a Drug's phenol moiety can react with the Linker, existing asan alcohol, under Mitsunobu conditions.

Typically, peptide-based Drugs can be prepared by forming a peptide bondbetween two or more amino acids and/or peptide fragments. Such peptidebonds can be prepared, for example, according to the liquid phasesynthesis method (see E. Schröder and K. Lübke, “The Peptides”, volume1, pp 76-136, 1965, Academic Press) that is well known in the field ofpeptide chemistry.

In one embodiment, a Drug is prepared by combining about astoichiometric equivalent of a dipeptide and a tripeptide, preferably ina one-pot reaction under suitable condensation conditions. This approachis illustrated in the following Schemes 5-7. Thus, the tripeptide 6 canbe prepared as shown in Scheme 5, and the dipeptide 9 can be prepared asshown in Scheme 6. The two fragments 6 and 9 can be condensed to providea Drug 10 as shown in Scheme 7.

The synthesis of an illustrative Stretcher having an electrophilicmaleimide group is illustrated in Schemes 8-9. General synthetic methodsuseful for the synthesis of a Linker are described in Scheme 10. Scheme11 shows the construction of a Linker unit having a val-cit group, anelectrophilic maleimide group and a PAB self-immolative Spacer group.Scheme 12 depicts the synthesis of a Linker having a phe-lys group, anelectrophilic maleimide group, with and without the PAB self-immolativeSpacer group. Scheme 13 presents a general outline for the synthesis ofa Drug-Linker Compound, while Scheme 14 presents an alternate route forpreparing a Drug-Linker Compound. Scheme 15 depicts the synthesis of abranched linker containing a BHMS group. Scheme 16 outlines theattachment of a Ligand to a Drug-Linker Compound to form aDrug-Linker-Ligand Conjugate, and Scheme 17 illustrates the synthesis ofDrug-Linker-Ligand Conjugates having 2 or 4 drugs per Ligand.

As illustrated in Scheme 5, a protected amino acid 1 (where PGrepresents an amine protecting group, R⁴ is selected from hydrogen,C₁-C₈ alkyl, C₃-C₈ carbocycle, —O—(C₁-C₈ alkyl), -aryl, alkyl-aryl,alkyl-(C₃-C₈ carbocycle), C₃-C₈ heterocycle, alkyl-(C₃-C₈ heterocycle)wherein R⁵ is selected from H and methyl; or R⁴ and R⁵ join, have theformula —(CR^(a)R^(b))_(n)— wherein R^(a) and R^(b) are independentlyselected from hydrogen, C₁-C₈ alkyl and C₃-C₈ carbocycle and n isselected from 2, 3, 4, 5 and 6, and form a ring with the carbon atom towhich they are attached) is coupled to t-butyl ester 2 (where R⁶ isselected from —H and —C₁-C₈ alkyl; and R⁷ is selected from hydrogen,C₁-C₈ alkyl, C₃-C₈ carbocycle, —O—(C₁-C₈ alkyl), -aryl, alkyl-aryl,alkyl-(C₃-C₈ carbocycle), C₃-C₈ heterocycle and alkyl-(C₃-C₈heterocycle)) under suitable coupling conditions, e.g., in the presenceof PyBrop and diisopropylethylamine, or using DCC (see, for example,Miyazaki, K. et. al. Chem. Pharm. Bull. 1995, 43(10), 1706-1718).

Suitable protecting groups PG, and suitable synthetic methods to protectan amino group with a protecting group are well known in the art. See,e.g., Greene, T. W. and Wuts, P. G. M., Protective Groups in OrganicSynthesis, 2nd Edition, 1991, John Wiley & Sons. Preferred protectedamino acids 1 are PG-Ile and, particularly, PG-Val, while other suitableprotected amino acids include, without limitation: PG-cyclohexylglycine,PG-cyclohexylalanine, PG-aminocyclopropane-1-carboxylic acid,PG-aminoisobutyric acid, PG-phenylalanine, PG-phenylglycine, andPG-tert-butylglycine. Z is a preferred protecting group. Fmoc is anotherpreferred protecting group. A preferred t-butyl ester 2 is dolaisoleuinet-butyl ester.

The dipeptide 3 can be purified, e.g., using chromatography, andsubsequently deprotected, e.g., using H₂ and 10% Pd—C in ethanol when PGis benzyloxycarbonyl, or using diethylamine for removal of an Fmocprotecting group. The resulting amine 4 readily forms a peptide bondwith an amino acid 5 (where R¹ is selected from —H, —C₁-C₈ alkyl and—C₃-C₈ carbocycle; and R² is selected from —H and —C₁-C₈ alkyl; or R¹and R² join, have the formula —(CR^(a)R^(b))_(n)— wherein R^(a) andR^(b) are independently selected from —H, —C₁-C₈ alkyl and —C₃-C₈carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring withthe nitrogen atom to which they are attached; and R³ is selected fromhydrogen, C₁-C₈ alkyl, C₃-C₈ carbocycle, —O—(C₁-C₈ alkyl), -aryl,alkyl-aryl, alkyl-(C₃-C₈ carbocycle), C₃-C₈ heterocycle and alkyl-(C₃-C₈heterocycle)). N,N-Dialkyl amino acids are preferred amino acids 5, suchas commercially available N,N-dimethyl valine. Other N,N-dialkyl aminoacids can be prepared by reductive bis-alkylation using known procedures(see, e.g., Bowman, R. E, Stroud, H. H J. Chem. Soc., 1950, 1342-1340).Fmoc-Me-L-Val and Fmoc-Me-L-glycine are two preferred amino acids 5useful for the synthesis of N-monoalkyl derivatives. The amine 4 and theamino acid 5 react to provide the tripeptide 6 using coupling reagentDEPC with triethylamine as the base.

Illustrative DEPC coupling methodology and the PyBrop couplingmethodology shown in Scheme 5 are outlined below in General Procedure Aand General Procedure B, respectively. Illustrative methodology for thedeprotection of a Z-protected amine via catalytic hydrogenation isoutlined below in General Procedure C.

General Procedure A: Peptide synthesis using DEPC. The N-protected orN,N-disubstituted amino acid or peptide 4 (1.0 eq.) and an amine 5 (1.1eq.) are diluted with an aprotic organic solvent, such asdichloromethane (0.1 to 0.5 M). An organic base such as triethylamine ordiisopropylethylamine (1.5 eq.) is then added, followed by DEPC (1.1eq.). The resulting solution is stirred, preferably under argon, for upto 12 hours while being monitored by HPLC or TLC. The solvent is removedin vacuo at room temperature, and the crude product is purified using,for example, HPLC or flash column chromatography (silica gel column).Relevant fractions are combined and concentrated in vacuo to affordtripeptide 6 which is dried under vacuum overnight.

General procedure B: Peptide synthesis using PyBrop. The amino acid 2(1.0 eq.), optionally having a carboxyl protecting group, is dilutedwith an aprotic organic solvent such as dichloromethane or DME toprovide a solution of a concentration between 0.5 and 1.0 mM, thendiisopropylethylamine (1.5 eq.) is added. Fmoc-, or Z-protected aminoacid 1 (1.1 eq.) is added as a solid in one portion, then PyBrop (1.2eq.) is added to the resulting mixture. The reaction is monitored by TLCor HPLC, followed by a workup procedure similar to that described inGeneral Procedure A.

General procedure C: Z-removal via catalytic hydrogenation. Z-protectedamino acid or peptide 3 is diluted with ethanol to provide a solution ofa concentration between 0.5 and 1.0 mM in a suitable vessel, such as athick-walled round bottom flask. 10% palladium on carbon is added (5-10%w/w) and the reaction mixture is placed under a hydrogen atmosphere.Reaction progress is monitored using HPLC and is generally completewithin 1-2 h. The reaction mixture is filtered through a pre-washed padof celite and the celite is again washed with a polar organic solvent,such as methanol after filtration. The eluent solution is concentratedin vacuo to afford a residue which is diluted with an organic solvent,preferably toluene. The organic solvent is then removed in vacuo toafford the deprotected amine 4.

Table 1 lists representative examples of tripeptide intermediates(compounds 39-43) that were prepared according to Scheme 5.

TABLE 1

Compound X¹ X² 39 Fmoc-N-Me-L-val L-val 40 Fmoc-N-Me-L-val L-ile 41Fmoc-N-Me-gly L-ile 42 dov L-val 43 dov L-ile ^(a)dov =N,N-dimethyl-L-valine

The dipeptide 9 can be readily prepared by condensation of the modifiedamino acid Boc-Dolaproine 7 (see, for example, Pettit, G. R., et al.Synthesis, 1996, 719-725), with (1S,2R)-norephedrine, L- orD-phenylalaninol, or with synthetic p-acetylphenethylamine 8 (U.S. Pat.No. 3,445,518 to Shavel et al.) using condensing agents well known forpeptide chemistry, such as, for example, DEPC in the presence oftriethylamine, as shown in Scheme 6. Compound 7 may also be condensedwith commercially available compounds in this manner to form dipeptidesof formula 9. Examples of commercially available compounds useful forthis purpose include, but are not limited to, norephedrine, ephedrine,and stereoisomers thereof (Sigma-Sigma-Aldrich), L- or D-phenylalaninol(Sigma-Aldrich), 2-phenylethylamine (Sigma-Aldrich),2-(4-aminophenyl)ethylamine (Sigma-Aldrich),1,2-ethanediamine-1,2-diphenyl (Sigma-Aldrich), or4-(2-aminoethyl)phenol (Sigma-Aldrich), or with synthetically preparedp-acetylphenethylamine, aryl- and heterocyclo-amides of L-phenylalanine,1-azidomethyl-2-phenylethylamine (prepared from phenylalaninol accordingto a general procedure described in J. Chem. Research (S), 1992, 391),and 1-(4-hydroxyphenyl)-2-phenylethylamine (European Patent PublicationNo. 0356035 A2) among others.

where R⁸ is independently selected from —H, —OH, —C₁-C₈ alkyl, —C₃-C₈carbocycle and —O—(C₁-C₈ alkyl); R⁹ is selected from —H and —C₁-C₈alkyl; and R¹⁰ is selected from:

where Z is —O—, —S—, —NH— or —N(R¹⁴)—; R¹¹is selected from —H, —OH,—NH₂, —NHR¹⁴, —N(R¹⁴)₂, —C₁-C₈ alkyl, —C₃-C₈ carbocycle, —O—(C₁-C₈alkyl), -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈ carbocycle),—C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈ heterocycle); or R¹¹ is anoxygen atom which forms a carbonyl unit (C═O) with the carbon atom towhich it is attached and a hydrogen atom on this carbon atom is replacedby one of the bonds in the (C═O) double bond; each R¹² is independentlyselected from -aryl and —C₃-C₈ heterocycle; R¹³ is selected from —H,—OH, —NH₂, —NHR¹⁴, —N(R¹⁴)₂, —C₁-C₈ alkyl, —C₃-C₈ carbocycle, —O—(C₁-C₈alkyl), -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈ carbocycle),—C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈ heterocycle); and each R¹⁴ isindependently —H or —C₁-C₈ alkyl.

Table 2 lists representative examples of dipeptides (Compounds 44-48)that were prepared according to Scheme 6.

TABLE 2

Compound Y 44

45

46

47

48

Scheme 7 illustrates a procedure useful for coupling tripeptide 6 anddipeptide 9 to form Drug 10. The coupling of 6 and 9 can be accomplishedusing a strong acid, e.g. TFA, to facilitate Boc and t-butyl estercleavage, from dipeptide 9 and tripeptide 6, respectively, followed bycondensation conditions, e.g., utilizing DEPC, or similar couplingreagent, in the presence of excess base (triethylamine or equivalent) toprovide Drug 10.

An illustrative procedure for the synthesis of Drug 10 as depicted inScheme 7 is outlined below in General Procedure D.

The R¹⁰ group of a Drug of general formula 10 can be further modified,if desired, to include a functional group that allows the drug to beattached to a Linker. Examples of useful modifications to the R¹⁰ groupof a Drug 10, include, but are not limited to the chemicaltransformations described below.

When R¹⁰ is

the hydroxyl group of R¹⁰ can be reacted with commercially available orsynthetically derived carboxylic acids or carboxylic acid derivatives,including but not limited to, carboxylic esters, acid chlorides,anhydrides and carbonates to provide the corresponding esters accordingto well known methods in the art. Coupling reagents, including, but notlimited to DCC/DMAP and EDCI/HOBt, can be useful in such couplingreactions between alcohols and carboxylic acids or carboxylic acidderivatives. In a preferred embodiment carboxylic acids are substitutedor unsubstituted aryl-carboxylic acids, for example, 4-aminobenzoicacid. Thus, condensation of a hydroxyl group of the R¹⁰ group shownabove with carboxylic acids provides drugs of the general structure 10

-   where R¹⁰ is

and where R¹¹, R¹², R¹⁴ and R¹⁵ are as previously described herein and Xis selected from —OH, —NH₂ and —NHR¹⁴.

When R¹⁰ is

the azido group of the drug can be reduced (for an example see J. Chem.Research (S), 1992, 391) to provide the corresponding amino derivativewherein R¹⁰ is

the amino group of which can be reacted with the carboxyl group of acarboxylic acid under general peptide coupling conditions to providedrugs of general structure 10, where

-   R¹⁰ is

and where R¹¹, R¹², R¹⁴ and R¹⁵ are as previously described herein and Xis selected from —OH, —NH₂ and —NHR¹⁴.

Carboxylic acids useful in the above regard include, but are not limitedto, 4-aminobenzoic acid, p-acetylbenzoic acid and2-amino-4-thiazolecarboxylic acid (Tyger Scientific, Inc., Ewing, N.J.).

An Fmoc-protected amino group may be present on an amine-containing R¹⁰group of Drug 10 (e.g., as depicted in Table 2). The Fmoc group isremovable from the protected amine using diethylamine (see GeneralProcedure E as an illustrative example described below).

General procedure D: Drug synthesis. A mixture of dipeptide 9 (1.0 eq.)and tripeptide 6 (1 eq.) is diluted with an aprotic organic solvent,such as dichloromethane, to form a 0.1M solution, then a strong acid,such as trifluoroacetic acid (½ v/v) is added and the resulting mixtureis stirred under a nitrogen atmosphere for two hours at 0° C. Thereaction can be monitored using TLC or, preferably, HPLC. The solvent isremoved in vacuo and the resulting residue is azeotropically driedtwice, preferably using toluene. The resulting residue is dried underhigh vacuum for 12 h and then diluted with and aprotic organic solvent,such as dichloromethane. An organic base such as triethylamine ordiisopropylethylamine (1.5 eq.) is then added, followed by either PyBrop(1.2 eq.) or DEPC (1.2 eq.) depending on the chemical functionality onthe residue. The reaction mixture is monitored by either TLC or HPLC andupon completion, the reaction is subjected to a workup procedure similaror identical to that described in General Procedure A.

General procedure E: Fmoc-removal using diethylamine. An Fmoc-protectedDrug 10 is diluted with an aprotic organic solvent such asdichloromethane and to the resulting solution is added diethylamine (½v/v). Reaction progress is monitored by TLC or HPLC and is typicallycomplete within 2 h. The reaction mixture is concentrated in vacuo andthe resulting residue is azeotropically dried, preferably using toluene,then dried under high vacuum to afford Drug 10 having a deprotectedamino group.

Thus, the above methods are useful for making Drugs that can be used inthe present invention.

To prepare a Drug-Linker Compound of the present invention, the Drug isreacted with a reactive site on the Linker. In general, the Linker canhave the structure:

when both a Spacer unit (—Y—) and a Stretcher unit (-A-) are present.Alternately, the Linker can have the structure:

when the Spacer unit (—Y—) is absent.

The Linker can also have the structure:

when both the Stretcher unit (-A-) and the Spacer unit (—Y—) are absent.

In general, a suitable Linker has an Amino Acid unit linked to anoptional Stretcher Unit and an optional Spacer Unit. Reactive Site 1 ispresent at the terminus of the Spacer and Reactive site 2 is present atthe terminus of the Stretcher. If a Spacer unit is not present, thenReactive site 1 is present at the C-terminus of the Amino Acid unit.

In one embodiment of the invention, Reactive Site No. 1 is reactive to anitrogen atom of the Drug, and Reactive Site No. 2 is reactive to asulfhydryl group on the Ligand. Reactive Sites 1 and 2 can be reactiveto different functional groups.

In one aspect of the invention, Reactive Site No. 1 is

In another aspect of the invention, Reactive Site No. 1 is

wherein R is —Br, —Cl, —O-Su or —O-(4-nitrophenyl).

In one embodiment, Reactive Site No. 1 is

wherein R is —Br, —Cl, —O-Su or —O-(4-nitrophenyl), when a Spacer unit(—Y—) is absent.

Linkers having

at Reactive Site No. 1 where R is —Br or —Cl can be prepared fromLinkers having

at Reactive Site No. 1 by reacting the —COOH group with PX₃ or PX₅,where X is —Br or —Cl. Alternatively, linkers having

at Reactive Site No. 1 can be prepared from Linkers having

at Reactive Site No. 1 by reacting the —COOH group with thionylchloride. For a general discussion of the conversion of carboxylic acidsto acyl halides, see March, Advanced Organic Chemistry—Reactions,Mechanisms and Structure, 4^(th) Ed., 1992, John Wiley and Sons, NewYork, p. 437-438.

In another aspect of the invention, Reactive Site No. 1 is

In still another aspect of the invention, Reactive Site No. 1 is

wherein R is —Cl, —O—CH(Cl)CCl₃ or —O-(4-nitrophenyl).

Linkers having

at Reactive Site No. 1 can be prepared from Linkers having

at Reactive Site No. 1 by reacting the —OH group with phosgene ortriphosgene to form the corresponding chloroformate. Linkers having

at Reactive Site No. 1 where R is —O—CH(Cl)CCl₃ or —O-(4-nitrophenyl)can be prepared from Linkers having

at Reactive Site No. 1 by reacting the —OC(O)Cl group with HO—CH(Cl)CCl₃or HO-(4-nitrophenyl), respectively. For a discussion of this chemistry,see March, Advanced Organic Chemistry—Reactions, Mechanisms andStructure, 4^(th) Ed., 1992, John Wiley and Sons, New York, p. 392.

In a further aspect of the invention, Reactive Site No. 1 is

wherein X is —F, —Cl, —Br, —I, or a leaving group such as —O-mesyl,—O-tosyl or —O-triflate.

Linkers having

at Reactive Site No. 1 where X is —O-mesyl, —O-tosyl and O-triflate canbe prepared from Linkers having

at Reactive Site No. 1 by reacting the —OH group with various reagents,including HCl, SOCl₂, PCl₅, PCl₃ and POCl₃ (where X is Cl); HBr, PBr₃,PBr₅ and SOBr₂ (where X is Br); HI (where X is I); and CH₃CH₂NSF₃(DAST), SF₄, SeF₄ and p-toluenesulfonyl fluoride (where X is F). For ageneral discussion on the conversion of alcohols to alkyl halides, seeMarch, Advanced Organic Chemistry—Reactions, Mechanisms and Structure,4^(th) Ed., 1992, John Wiley and Sons, New York, p. 431-433.

Linkers having

at Reactive Site No. 1 where X is —O-mesyl, —O-tosyl and —O-triflate,can be prepared from Linkers having

at Reactive Site No. 1 by reacting the —OH group with variousmesylating, tosylating and triflating reagents, respectively. Suchreagents and methods for their use will be well known to one of ordinaryskill in the art of organic synthesis. For a general discussion ofmesyl, tosyl and triflates as leaving groups, see March, AdvancedOrganic Chemistry—Reactions, Mechanisms and Structure, 4^(th) Ed., 1992,John Wiley and Sons, New York, p. 353-354.

In one embodiment, when a Spacer unit (—Y—) is present, Reactive SiteNo. 1 is

wherein R is —Cl, —O—CH(Cl)CCl₃ or —O-(4-nitrophenyl) and X is —F, —Cl,—Br, —I, or a leaving group such as —O-mesyl, —O-tosyl or —O-triflate.

In another aspect of the invention, Reactive Site No. 1 is

In still another aspect of the invention, Reactive Site No. 1 is ap-nitrophenyl carbonate having the formula

In one aspect of the invention, Reactive Site No. 2 is a thiol-acceptinggroup. Suitable thiol-accepting groups include haloacetamide groupshaving the formula

where X represents a leaving group, preferably O-mesyl, O-tosyl, —Cl,—Br, or —I; or a maleimide group having the formula

Useful Linkers can be obtained via commercial sources, such as MolecularBiosciences Inc. (Boulder, Colo.), or synthesized in accordance withprocedures described in U.S. Pat. No. 6,214,345 to Firestone et al.,summarized in Schemes 8-10 below.

where X is —CH₂— or —CH₂OCH₂—; and n is an integer ranging either from0-10 when X is —CH₂—; or 1-10 when X is —CH₂OCH₂—.

The method shown in Scheme 9 combines maleimide with a glycol underMitsunobu conditions to make a polyethylene glycol maleimide Stretcher(see for example, Walker, M. A. J. Org. Chem. 1995, 60, 5352-5),followed by installation of a p-nitrophenyl carbonate Reactive Sitegroup.

where E is —CH₂— or —CH₂OCH₂—; and e is an integer ranging from 0-8;

Alternatively, PEG-maleimide and PEG-haloacetamide stretchers can beprepared as described by Frisch, et al., Bioconjugate Chem. 1996, 7,180-186.

Scheme 10 illustrates a general synthesis of an illustrative Linker unitcontaining a maleimide Stretcher group and optionally a p-aminobenzylether self-immolative Spacer.

-   -   where Q is —C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -halogen,-nitro or        -cyano; m is an integer ranging from 0-4; and n is an integer        ranging from 0-10.

Useful Stretchers may be incorporated into a Linker using thecommercially available intermediates from Molecular Biosciences(Boulder, Colo.) described below by utilizing known techniques oforganic synthesis.

Stretchers of formula (IIIa) can be introduced into a Linker by reactingthe following intermediates with the N-terminus of an Amino Acid unit asdepicted in Schemes 11 and 12:

where n is an integer ranging from 1-10 and T is —H or —SO₃Na;

where n is an integer ranging from 0-3;

Stretcher units of formula (IIIb) can be introduced into a Linker byreacting the following intermediates with the N-terminus of an AminoAcid unit:

-   -   where X is —Br or —I; and

Stretcher units of formula (IV) can be introduced into a Linker byreacting the following intermediates with the N-terminus of an AminoAcid unit:

Stretcher units of formula (Va) can be introduced into a Linker byreacting the following intermediates with the N-terminus of an AminoAcid unit:

Other Stretchers useful in the invention may be synthesized according toknown procedures. Aminooxy Stretchers of the formula shown below can beprepared by treating alkyl halides with N-Boc-hydroxylamine according toprocedures described in Jones, D. S. et al., Tetrahedron Letters, 2000,41(10), 1531-1533; and Gilon, C. et al., Tetrahedron, 1967, 23(11),4441-4447.

where —R¹⁷— is selected from —C₁-C₁₀ alkylene-, —C₃-C₈ carbocyclo-,—O—(C₁-C₈ alkyl)-, -arylene-, —C₁-C₁₀ alkylene-arylene-, -arylene-C₁-C₁₀alkylene-, —C₁-C₁₀ alkylene-(C₃-C₈ carbocyclo)-, —(C₃-C₈carbocyclo)-C₁-C₁₀ alkylene-, —C₃-C₈ heterocyclo-, —C₁-C₁₀alkylene-(C₃-C₈ heterocyclo)-, —(C₃-C₈ heterocyclo)-C₁-C₁₀ alkylene-,—(CH₂CH₂O)_(r)—, —(CH₂CH₂O)_(r)—CH₂—; and r is an integer ranging from1-10;

Isothiocyanate Stretchers of the formula shown below may be preparedfrom isothiocyanatocarboxylic acid chlorides as described in Angew.Chem., 1975, 87(14), 517.

where —R¹⁷— is as described herein.

Scheme 11 shows a method for obtaining of a val-cit dipeptide Linkerhaving a maleimide Stretcher and optionally a p-aminobenzylself-immolative Spacer.

where Q is —C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -halogen, -nitro or -cyano;and m is an integer ranging from 0-4.

Scheme 12 illustrates the synthesis of a phe-lys(Mtr) dipeptide Linkerunit having a maleimide Stretcher unit and a p-aminobenzylself-immolative Spacer unit. Starting material 23 (lys(Mtr)) iscommercially available (Bachem, Torrance, Calif.) or can be preparedaccording to Dubowchik, et al. Tetrahedrom Letters 1997, 38, 5257-60.

where Q is —C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -halogen, -nitro or -cyano;and m is an integer ranging from 0-4.

As shown in Scheme 13, a Linker can be reacted with an amino group of aDrug 10 to form a Drug-Linker Compound that contains an amide orcarbamate group, linking the Drug unit to the Linker unit. When ReactiveSite No. 1 is a carboxylic acid group, as in Linker 29, the couplingreaction can be performed using HATU or PyBrop and an appropriate aminebase, resulting in a Drug-Linker Compound 30, containing a amide bondbetween the Drug unit and the Linker unit. When Reactive Site No. 1 is acarbonate, as in Linker 31, the Linker can be coupled to the Drug usingHOBt in a mixture of DMF/pyridine to provide a Drug-Linker Compound 32,containing a carbamate bond between the Drug unit and the Linker unit.

When Reactive Site No. 1 is an hydroxyl group, such as Linker 33, theLinker can be coupled with a phenol group of a Drug using Mitsunobuchemistry to provide a Drug-Linker Compound 34 having an ether linkagebetween the Drug unit and the Linker unit.

Alternately, when Reactive Site No. 1 is a good leaving group, such asin Linker 70, the Linker can be coupled with a hydroxyl group or anamine group of a Drug via a nucleophilic substitution process to providea Drug-Linker Compound having an ether linkage (34) or an amine linkage(71) between the Drug unit and the Linker unit.

Illustrative methods useful for linking a Drug to a Ligand to form aDrug-Linker Compound are depicted in Scheme 13 and are outlined inGeneral Procedures G-J.

General Procedure G: Amide formation using HATU. A Drug 10 (1.0 eq.) andan N-protected Linker containing a carboxylic acid Reactive site (1.0eq.) are diluted with a suitable organic solvent, such asdichloromethane, and the resulting solution is treated with HATU (1.5eq.) and an organic base, preferably pyridine (1.5 eq.). The reactionmixture is allowed to stir under an inert atmosphere, preferably argon,for 6 h, during which time the reaction mixture is monitored using HPLC.The reaction mixture is concentrated and the resulting residue ispurified using HPLC to yield the amide 30.

General Procedure H: Carbamate formation using HOBt. A mixture of aLinker 31 having a p-nitrophenyl carbonate Reactive site (1.1 eq.) andDrug 10 (1.0 eq.) are diluted with an aprotic organic solvent, such asDMF, to provide a solution having a concentration of 50-100 mM, and theresulting solution is treated with HOBt (2.0 eq.) and placed under aninert atmosphere, preferably argon. The reaction mixture is allowed tostir for 15 min, then an organic base, such as pyridine (¼ v/v), isadded and the reaction progress is monitored using HPLC. The Linker istypically consumed within 16 h. The reaction mixture is thenconcentrated in vacuo and the resulting residue is purified using, forexample, HPLC to yield the carbamate 32.

General Procedure I: Ether formation using Mitsunobu chemistry. A Drugof general formula 10, which contains a free hydroxyl group, is dilutedwith THF to make a 1.0 M solution and to this solution is added a Linker(1.0 eq) containing an hydroxy group at Reactive site No. 1 (33),followed by triphenylphosphine (1.5 eq.). The reaction mixture is putunder an argon atmosphere and cooled to 0° C. DEAD (1.5 eq.) is thenadded dropwise via syringe and the reaction is allowed to stir at roomtemperature while being monitored using HPLC. The reaction is typicallycomplete in 0.5-12 h, depending on the substrates. The reaction mixtureis diluted with water (in volume equal to that of the THF) and thereaction mixture is extracted into EtOAc. The EtOAc layer is washedsequentially with water and brine, then dried over MgSO4 andconcentrated. The resulting residue is purified via flash columnchromatography using a suitable eluent to provide ether 34.

General Procedure J: Ether/amine Formation via NucleophilicSubstitution. A Drug of general formula 10, which contains a freehydroxyl group or a free amine group, is diluted with a polar aproticsolvent, such as THF, DMF or DMSO, to make a 1.0 M solution and to thissolution is added a non-nucleophilic base (about 1.5 eq), such aspyridine, diisopropylethylamine or triethylamine. The reaction mixtureis allowed to stir for about 1 hour, and to the resulting solution isadded an approximately 1.0M solution of Linker 70 in a polar aproticsolvent, such as THF, DMF or DMSO. The resulting reaction is stirredunder an inert atmosphere while being monitored using TLC or HPLC. Thereaction is typically complete in 0.5-12 h, depending on the substrates.The reaction mixture is diluted with water (in volume equal to that ofthe reaction volume) and extracted into EtOAc. The EtOAc layer is washedsequentially with water, 1N HCl, water, and brine, then dried over MgSO4and concentrated. The resulting residue is purified via flash columnchromatography using a suitable eluent to provide an ether of formula 34or an amine of formula 71, depending on whether the drug 10 contained afree hydroxyl group or a free amine group.

An alternate method of preparing Drug-Linker Compounds of the inventionis outlined in Scheme 14. Using the method of Scheme 14, the Drug isattached to a partial Linker unit (19a, for example), which does nothave a Stretcher unit attached. This provides intermediate 35, which hasan Amino Acid unit having an Fmoc-protected N-terminus. The Fmoc groupis then removed and the resulting amine intermediate 36 is then attachedto a Stretcher unit via a coupling reaction catalyzed using PyBrop orDEPC. The construction of Drug-Linker Compounds containing either abromoacetamide Stretcher 39 or a PEG maleimide Stretcher 38 isillustrated in Scheme 14.

where Q is —C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -halogen, -nitro or -cyano;and m is an integer ranging from 0-4.

Methodology useful for the preparation of a Linker unit containing abranched spacer is shown in Scheme 15.

Scheme 15 illustrates the synthesis of a val-cit dipeptide linker havinga maleimide 3 Stretcher unit and a bis(4-hydroxymethyl)styrene (BHMS)unit. The synthesis of the

BHMS intermediate (75) has been improved from previous literatureprocedures (see International Publication No, WO 9813059 to Firestone etal., and Crozet, M. P.; Archaimbault, G.; Vanelle, P.; Nouguier, R.Tetrahedron Lett. 1985, 26, 5133-5134) and utilizes as startingmaterials, commercially available diethyl(4-nitrobenzyl)phosphonate (72)and commercially available 2,2-dimethyl-1,3-dioxan-5-one (73). Linkers77 and 79 can be prepared from intermediate 75 using the methodologydescribed in Scheme 11.

Scheme 16 illustrates methodology useful for making Drug-Linker-Ligandconjugates of the invention having about 2 to about 4 drugs perantibody.

General Procedure K: Preparation of Conjugates having about 2 to about 4drugs per antibody.

Partial Reduction of the Antibody

In general, to prepare conjugates having 2 drugs per antibody, therelevant antibody is reduced using a reducing agent such asdithiothreitol (DTT) or tricarbonyl ethylphosphine (TCEP) (about 1.8equivalents) in PBS with 1 mM DTPA, adjusted to pH 8 with 50 mM borate.The solution is incubated at 37° C. for 1 hour, purified using a 50 mlG25 desalting column equilibrated in PBS/1 mM DTPA at 4° C. The thiolconcentration can be determined according to General Procedure M, theprotein concentration can be determined by dividing the A280 value by1.58 extinction coefficient (mg/ml), and the ratio of thiol to antibodycan be determined according to General Procedure N.

Conjugates having 4 drugs per antibody can be made using the samemethodology, using about 4.2 equivalents of a suitable reducing agent topartially reduce the antibody.

Conjugation of Drug-Linker to Partially Reduced Antibody

The partially reduced antibody samples can be conjugated to acorresponding Drug-Linker compound using about 2.4 and about 4.6 molarequivalents of Drug-Linker compound per antibody to prepare the 2 and 4drug per antibody conjugates, respectively. The conjugation reactionsare incubated on ice for 1 hour, quenched with about 20-fold excess ofcysteine to drug, and purified by elution over a G25 desalting column atabout 4° C. The resulting Drug-Linker-Ligand conjugates are concentratedto about 3 mg/ml, sterile filtered, aliquoted and stored frozen.

Scheme 17 depicts the construction of a Drug-Linker-Ligand Conjugate byreacting the sulfhydryl group of a Ligand with a thiol-acceptor group onthe Linker group of a Drug-Linker Compound.

Illustrative methods for attaching a Ligand antibody to a Drug-LinkerCompound are outlined below in General Procedures L-R.

General Procedure L: Attachment of an Antibody Ligand to a Drug-LinkerCompound. All reaction steps are typically carried out at 4° C. Wherethe Ligand is a monoclonal antibody having one or more disulfide bonds,solutions of the monoclonal antibody (5-20 mg/mL) in phosphate bufferedsaline, pH 7.2, are reduced with dithiothreitol (10 mM final) at 37° C.for 30 minutes (See General Procedure M) and separation of low molecularweight agents is achieved by size exclusion chromatography on SephadexG25 columns in PBS containing 1 mM diethylenetriaminepentaacetic acid.

The sulfhydryl content in the Ligand can be determined using5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) as described in GeneralProcedure M (see Riddles, P. W., Blakeley, R. L., and Zerner, B. (1979)Anal. Biochem. 94, 75-81). To a PBS solution of Ligand reduced accordingto General Procedure L, a Drug-Linker Compound in MeCN is added so thatthe solution is 20% MeCN/PBS (vol/vol). The amount of Drug-LinkerCompound is approximately 10% more than the total number of sulfhydrylgroups on a Ligand. After 60 min at 4° C., cysteine is added (20-foldexcess over concentration of the Drug-Linker Compound), the solution isconcentrated by ultrafiltration, and any low molecular weight agents areremoved by gel filtration. The number of Drug-Linker Compounds perantibody is determined by uv/vis spectroscopy using formulas derivedfrom the relative extinction coefficients of the Ligands and Drug-LinkerCompounds as described in General Procedure O. The amount of quenchedDrug-Linker Compound is then determined as described in GeneralProcedure P using reverse-phase HPLC. The aggregation state of theLigand Antibodies of the Drug-Linker-Ligand Conjugates can be determinedusing size-exclusion HPLC as described in General Procedure R. TheDrug-Linker-Ligand Conjugates can be used without further purification.

General Procedure M: Reduction of the interchain disulfide bonds of anAntibody. To a solution of 24 mg of an antibody (2.4 mL of 10 mg/mLsolution) in suitable buffer is added 300 μL of Borate buffer (500 mMsodium borate/500 mM sodium chloride, pH 8.0) followed by 300 μL ofDithiothreitol (DTT, 100 mM solution in H₂O). The reaction mixture isstirred using a vortex instrument and incubated at 37° C. for 30 min.Three PD10 columns are equilibrated with PBS containing 1 mM DTPA (inPBS) and the reduced antibody is eluted through the three PD10 columnsand collected in 4.2 mL PBS/DTPA solution (1.4 mL per column). Thereduced antibody is then stored on ice. The number of thiols perantibody and the antibody concentration are determined according toGeneral Procedure N.

General Procedure N: Determination of Number of Thiols Per Ligand

A reference sample of a Ligand or a sample of an antibody reducedaccording to General Procedure L is diluted to about 1:40 (w/w) in PBS,and the uv absorbance of the solution is measured at 280 nm usingstandard uv spectroscopic methods. Preferably, the ratio of Ligand:PBSin the solution is such that the uv absorbance ranges from about0.13-0.2 AU (absorbance units).

A test sample of a Ligand or a test sample of an antibody reducedaccording to General Procedure L is diluted to about 1:20 with a PBSsolution containing about 15 μL DTNB stock solution/mL PBS. A blanksample containing DTNB at the same concentration as the test solution(i.e., 15 μL DTNB stock/mL PBS) is then prepared. The spectrophotometeris referenced at zero nm with the blank sample, then the absorbance ofthe test sample is measured at 412 nm.

The molar concentration of the antibody is then determined using theformula: [Ligand]=(OD₂₈₀/2.24e⁵)×dilution factor.

The molar concentration of thiol is then determined using the formula:[—SH]=(OD₄₁₂/1.415e⁴)×dilution factor.

The [SH]/[Ligand] ratio is then calculated. A reduced monoclonalantibody Ligand can have from 1 to about 20 sulfhydryl groups, buttypically has between about 6 to about 9 sulfhydryl groups. In apreferred embodiment, the [SH]/[Ligand] ratio range is from about 7 toabout 9.

It is understood that the [SH]/[Ligand] ratio is the average number of-A_(a)-W_(w)—Y_(y)-D units per Ligand unit.

General Procedure O: Determination of the number of Drug molecules perAntibody in a Drug-Linker-Antibody Conjugate. The Drug:Antibody ratiofor a Drug-Linker-Antibody Conjugate is determined by measuring thenumber of Dithiothreitol (DTT) reducible thiols that remain afterconjugation, using the following method: A 200 mL sample of aDrug-Linker-Antibody conjugate is treated with DTT (100 mM solution inwater) to bring the concentration to 10 mM DTT. The resulting solutionis incubated at 37° C. for 30 min, then eluted through a PD10 columnusing PBS/DTPA as the eluent. The OD₂₈₀ of the reduced conjugate is thenmeasured and the molar concentration is measured according to GeneralProcedure Q.

The molar concentration of thiol is determined using DTNB as describedin General Procedure M. The ratio of thiol concentration to antibodyconcentration is then calculated and the Drug:Ligand ratio is thedifference between the Thiol:Antibody ratio (determined using GeneralProcedure N) and the Drug:Antibody ratio as determined in the previousparagraph.

General Procedure P: Determination of the amount of quenched Drug-Linkercompound in a Drug-Linker-Antibody Conjugate. This assay provides aquantitative determination of the Drug-Linker in theDrug-Linker-Antibody conjugate that is not covalently bound to Antibody.Assuming that all maleimide groups of Drug-Linker in the reactionmixture have been quenched with Cysteine, the unbound drug is theCysteine quenched adduct of the Drug-Linker Compound, i.e.Drug-Linker-Cys. The proteinaceous Drug-Linker-Antibody Conjugate isdenatured, precipitated, and isolated by centrifugation under conditionsin which the Drug-Linker-Cys is soluble. The unbound Drug-Linker-Cys isdetected quantitatively by HPLC, and the resulting chromatogram iscompared to a standard curve to determine the concentration of unboundDrug-Linker-Cys in the sample. This concentration is divided by thetotal concentration of Drug in the conjugate as determined using GeneralProcedure 0 and General Procedure Q.

Specifically, 100 mL of a 100 μM Drug-Linker-Cys adduct “workingsolution” is prepared by adding 1 μL of 100 mM Cysteine in PBS/DTPA andan appropriate volume of stock solution of a Drug-Linker compound to 98μL of 50% methanol/PBS. The “appropriate volume” in liters is calculatedusing the formula: V=1c-8/[Drug-Linker]. Six tubes are then labelled asfollows: “0”, “0.5”, “1”, “2”, “3”, and “5”, and appropriate amounts ofworking solution are placed in each tube and diluted with 50%methanol/PBS to give a total volume of 100 mL in each tube. The labelsindicate the μM concentration of the standards.

A 50 μL solution of a Drug-Linker-Antibody Conjugate and a 50 μLsolution of the Cysteine quenched reaction mixture (“qrm”) are collectedin separate test tubes and are each diluted with 50 μL, of methanol thathas been cooled to −20° C. The samples are then cooled to −20° C. over10 min.

The samples are then centrifuged at 13000 rpm in a desktop centrifugefor 10 min. The supernatants are transferred to HPLC vials, and 90 μLaliquots of each sample are separately analyzed using HPLC (C12 RPcolumn (Phenomenex); monitored at the absorbance maximum of theDrug-Linker Compound using a flow rate of 1.0 mL/min. The eluent used isa linear gradient of MeCN ranging from 10 to 90% in aqueous 5 mMammonium phosphate, pH 7.4, over 10 min; then 90% MeCN over 5 min.; thenreturning to initial conditions). The Drug-Linker-Cys adduct typicallyelutes between about 7 and about 10 minutes.

A standard curve is then prepared by plotting the Peak Area of thestandards vs. their concentration (in μM). Linear regression analysis isperformed to determine the equation and correlation coefficient of thestandard curve. R2 values are typically >0.99. From the regressionequation is determined the concentration of the Drug-Linker-Cys adductin the HPLC sample and in the conjugate, using the formulas:

[Drug-Linker-Cys]_((HPLC spl))=(Peak area−intercept)/slope;

[Drug-Linker-Cys]_((conjugate))=2×[Drug-Linker-Cys]_((HPLC spl))

The percent of Drug-Linker-Cys adduct present can be determined usingthe formula:

% Drug-Linker-Cys=100×[Drug-Linker-Cys]_((conjugate))/[drug]

where [drug]=[Conjugate]×drug/Ab, [Conjugate] is determined using theconjugate concentration assay, and the Drug: Antibody ratio isdetermined using the Drug: Antibody ratio assay.

General Procedure Q: Determination of Drug-Linker-Antibody Conjugateconcentration for drug linkers with minimal uv absorbance at 280 nm. Theconcentration of Drug-Linker-Antibody conjugate can be determined in thesame manner for the concentration of the parent antibody, by measuringthe absorbance at 280 nm of an appropriate dilution, using the followingformula:

[Conjugate](mg/mL)=(OD₂₈₀×dilution factor/1.4)×0.9

Determination of Drug-Linker-Antibody Conjugate concentration for druglinkers with substantial uv absorbance at 280 nm (e.g. Compounds 68 and69). Because the absorbances of Compounds 68 and 69 overlap with theabsorbances of an antibody, spectrophotometric determination of theconjugate concentration is most useful when the measurement is performedusing the absorbances at both 270 nm and 280 nm. Using this data, themolar concentration of Drug-Linker-Ligand conjugate is given by thefollowing formula:

[Conjugate]=(OD₂₈₀×1.23 e⁻⁵−OD₂₇₀×9.35e⁻⁶)×dilution factor where the1.23e⁻⁵ and 9.35e⁻⁶ are calculated from the molar extinctioncoefficients of the drug and the antibody, which are estimated as:

-   -   e₂₇₀ Drug=2.06e⁴ e₂₇₀ Antibody=1.87e⁵    -   e₂₈₀ Drug=1.57e⁴ e₂₈₀ Antibody=2.24e⁵

Determination of Drug-Linker-Antibody Conjugate concentration for druglinkers with substantial uv absorbance at 280 nm (e.g. Compounds 68 and69). Because the absorbances of Compounds 68 and 69 overlap with theabsorbances of an antibody, spectrophotometric determination of theconjugate concentration is most useful when the measurement is performedusing the absorbances at both 270 nm and 280 nm. Using this data, themolar concentration of Drug-Linker-Ligand conjugate is given by thefollowing formula:

[Conjugate]=(OD₂₈₀×1.23 e ⁻⁵−OD₂₇₀×9.35e ⁻⁶)×dilution factor where thevalues 1.23e⁻⁵ and 9.35e⁻⁶ are calculated from the molar extinctioncoefficients of the drug and the antibody, which are estimated as:

-   -   e₂₇₀ Drug=2.06e⁴ e₂₇₀ Antibody=1.87e⁵    -   e₂₈₀ Drug=1.57e⁴ e₂₈₀ Antibody=2.24e⁵

General Procedure R: Determination of the aggregation state of TheAntibody in a Drug-Linker-Antibody Conjugate. A suitable quantity (−10μg) of a Drug-Linker-Antibody Conjugate is eluted through asize-exclusion chromatography (SEC) column (Tosoh Biosep SW3000 4.6mm×30 cm eluted at 0.35 mL/min. with PBS) under standard conditions.Chromatograms are obtained at 220 nm and 280 nm and the OD₂₈₀/OD₂₂₀ratio is calculated. The corresponding aggregate typically has aretention time of between about 5.5 and about 7 min, and has about thesame OD₂₈₀/OD₂₂₀ ratio as the monomeric Drug-Linker-Antibody Conjugate.

Compositions

In other aspects, the present invention provides a compositioncomprising an effective amount of a Compound of the Invention and apharmaceutically acceptable carrier or vehicle. For convenience, theDrug units, Drug-Linker Compounds and Drug-Linker-Ligand Conjugates ofthe invention can simply be referred to as compounds of the invention.The compositions are suitable for veterinary or human administration.

The compositions of the present invention can be in any form that allowsfor the composition to be administered to an animal. For example, thecomposition can be in the form of a solid, liquid or gas (aerosol).Typical routes of administration include, without limitation, oral,topical, parenteral, sublingual, rectal, vaginal, ocular, andintranasal.

Parenteral administration includes subcutaneous injections, intravenous,intramuscular, intrasternal injection or infusion techniques.Preferably, the compositions are administered parenterally.Pharmaceutical compositions of the invention can be formulated so as toallow a Compound of the Invention to be bioavailable upon administrationof the composition to an animal. Compositions can take the form of oneor more dosage units, where for example, a tablet can be a single dosageunit, and a container of a Compound of the Invention in aerosol form canhold a plurality of dosage units.

Materials used in preparing the pharmaceutical compositions can benon-toxic in the amounts used. It will be evident to those of ordinaryskill in the art that the optimal dosage of the active ingredient(s) inthe pharmaceutical composition will depend on a variety of factors.Relevant factors include, without limitation, the type of animal (e.g.,human), the particular form of the Compound of the Invention, the mannerof administration, and the composition employed.

The pharmaceutically acceptable carrier or vehicle can be particulate,so that the compositions are, for example, in tablet or powder form. Thecarrier(s) can be liquid, with the compositions being, for example, anoral syrup or injectable liquid. In addition, the carrier(s) can begaseous, so as to provide an aerosol composition useful in, e.g.,inhalatory administration.

When intended for oral administration, the composition is preferably insolid or liquid form, where semi-solid, semi-liquid, suspension and gelforms are included within the forms considered herein as either solid orliquid.

As a solid composition for oral administration, the composition can beformulated into a powder, granule, compressed tablet, pill, capsule,chewing gum, wafer or the like form. Such a solid composition typicallycontains one or more inert diluents. In addition, one or more of thefollowing can be present: binders such as carboxymethylcellulose, ethylcellulose, microcrystalline cellulose, or gelatin; excipients such asstarch, lactose or dextrins, disintegrating agents such as alginic acid,sodium alginate, Primogel, corn starch and the like; lubricants such asmagnesium stearate or Sterotex; glidants such as colloidal silicondioxide; sweetening agents such as sucrose or saccharin, a flavoringagent such as peppermint, methyl salicylate or orange flavoring, and acoloring agent.

When the composition is in the form of a capsule, e.g., a gelatincapsule, it can contain, in addition to materials of the above type, aliquid carrier such as polyethylene glycol, cyclodextrin or a fatty oil.

The composition can be in the form of a liquid, e.g., an elixir, syrup,solution, emulsion or suspension. The liquid can be useful for oraladministration or for delivery by injection. When intended for oraladministration, a composition can comprise one or more of a sweeteningagent, preservatives, dye/colorant and flavor enhancer. In a compositionfor administration by injection, one or more of a surfactant,preservative, wetting agent, dispersing agent, suspending agent, buffer,stabilizer and isotonic agent can also be included.

The liquid compositions of the invention, whether they are solutions,suspensions or other like form, can also include one or more of thefollowing: sterile diluents such as water for injection, salinesolution, preferably physiological saline, Ringer's solution, isotonicsodium chloride, fixed oils such as synthetic mono or digylcerides whichcan serve as the solvent or suspending medium, polyethylene glycols,glycerin, cyclodextrin, propylene glycol or other solvents;antibacterial agents such as benzyl alcohol or methyl paraben;antioxidants such as ascorbic acid or sodium bisulfite; chelating agentssuch as ethylenediaminetetraacetic acid; buffers such as acetates,citrates or phosphates and agents for the adjustment of tonicity such assodium chloride or dextrose. A parenteral composition can be enclosed inampoule, a disposable syringe or a multiple-dose vial made of glass,plastic or other material. Physiological saline is a preferred adjuvant.An injectable composition is preferably sterile.

The amount of the Compound of the Invention that is effective in thetreatment of a particular disorder or condition will depend on thenature of the disorder or condition, and can be determined by standardclinical techniques. In addition, in vitro or in vivo assays canoptionally be employed to help identify optimal dosage ranges. Theprecise dose to be employed in the compositions will also depend on theroute of administration, and the seriousness of the disease or disorder,and should be decided according to the judgment of the practitioner andeach patient's circumstances.

The compositions comprise an effective amount of a Compound of theInvention such that a suitable dosage will be obtained. Typically, thisamount is at least about 0.01% of a Compound of the Invention by weightof the composition. When intended for oral administration, this amountcan be varied to range from about 0.1% to about 80% by weight of thecomposition. Preferred oral compositions can comprise from about 4% toabout 50% of the Compound of the Invention by weight of the composition.Preferred compositions of the present invention are prepared so that aparenteral dosage unit contains from about 0.01% to about 2% by weightof the Compound of the Invention.

For intravenous administration, the composition can comprise from about1 to about 250 mg of a Compound of the Invention per kg of the animal'sbody weight. Preferably, the amount administered will be in the rangefrom about 4 to about 25 mg/kg of body weight of the Compound of theInvention.

Generally, the dosage of Compound of the Invention administered to ananimal is typically about 0.1 mg/kg to about 250 mg/kg of the animal'sbody weight. Preferably, the dosage administered to an animal is betweenabout 0.1 mg/kg and about 20 mg/kg of the animal's body weight, morepreferably about 1 mg/kg to about 10 mg/kg of the animal's body weight.

The Compounds of the Invention or compositions can be administered byany convenient route, for example by infusion or bolus injection, byabsorption through epithelial or mucocutaneous linings (e.g., oralmucosa, rectal and intestinal mucosa, etc.). Administration can besystemic or local. Various delivery systems are known, e.g.,encapsulation in liposomes, microparticles, microcapsules, capsules,etc., and can be used to administer a Compound of the Invention orcomposition. In certain embodiments, more than one Compound of theInvention or composition is administered to an animal. Methods ofadministration include, but are not limited to, oral administration andparenteral administration; parenteral administration including, but notlimited to, intradermal, intramuscular, intraperitoneal, intravenous,subcutaneous; intranasal, epidural, sublingual, intranasal,intracerebral, intraventricular, intrathecal, intravaginal, transdermal,rectally, by inhalation, or topically to the ears, nose, eyes, or skin.The preferred mode of administration is left to the discretion of thepractitioner, and will depend in-part upon the site of the medicalcondition (such as the site of cancer or autoimmune disease).

In a preferred embodiment, the present Compounds of the Invention orcompositions are administered parenterally.

In a more preferred embodiment, the present Compounds of the Inventionor compositions are administered intravenously.

In specific embodiments, it can be desirable to administer one or moreCompounds of the Invention or compositions locally to the area in needof treatment. This can be achieved, for example, and not by way oflimitation, by local infusion during surgery; topical application, e.g.,in conjunction with a wound dressing after surgery; by injection; bymeans of a catheter; by means of a suppository; or by means of animplant, the implant being of a porous, non-porous, or gelatinousmaterial, including membranes, such as sialastic membranes, or fibers.In one embodiment, administration can be by direct injection at the site(or former site) of a cancer, tumor or neoplastic or pre-neoplastictissue. In another embodiment, administration can be by direct injectionat the site (or former site) of a manifestation of an autoimmunedisease.

In certain embodiments, it can be desirable to introduce one or moreCompounds of the Invention or compositions into the central nervoussystem by any suitable route, including intraventricular and intrathecalinjection. Intraventricular injection can be facilitated by anintraventricular catheter, for example, attached to a reservoir, such asan Ommaya reservoir.

Pulmonary administration can also be employed, e.g., by use of aninhaler or nebulizer, and formulation with an aerosolizing agent, or viaperfusion in a fluorocarbon or synthetic pulmonary surfactant. Incertain embodiments, the Compounds of the Invention or compositions canbe formulated as a suppository, with traditional binders and carrierssuch as triglycerides.

In another embodiment, the Compounds of the invention can be deliveredin a vesicle, in particular a liposome (see Langer, Science249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy ofInfectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss,New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; seegenerally ibid.)

In yet another embodiment, the Compounds of the Invention orcompositions can be delivered in a controlled release system. In oneembodiment, a pump can be used (see Langer, supra; Sefton, CRC Crit.Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980);Saudek et al., N. Engl. J. Med. 321:574 (1989)). In another embodiment,polymeric materials can be used (see Medical Applications of ControlledRelease, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974);Controlled Drug Bioavailability, Drug Product Design and Performance,Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J.Macromol. Sci. Rev. Macromol. Chem. 23:61(1983); see also Levy et al.,Science 228:190 (1985); During et al., Ann. Neurol. 25:351 (1989);Howard et al., J. Neurosurg. 71:105 (1989)). In yet another embodiment,a controlled-release system can be placed in proximity of the target ofthe Compounds of the Invention or compositions, e.g., the brain, thusrequiring only a fraction of the systemic dose (see, e.g., Goodson, inMedical Applications of Controlled Release, supra, vol. 2, pp. 115-138(1984)). Other controlled-release systems discussed in the review byLanger (Science 249:1527-1533 (1990)) can be used.

The term “carrier” refers to a diluent, adjuvant or excipient, withwhich a Compound of the Invention is administered. Such pharmaceuticalcarriers can be liquids, such as water and oils, including those ofpetroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil, sesame oil and the like. The carriers can besaline, gum acacia, gelatin, starch paste, talc, keratin, colloidalsilica, urea, and the like. In addition, auxiliary, stabilizing,thickening, lubricating and coloring agents can be used. In oneembodiment, when administered to an animal, the Compounds of theInvention or compositions and pharmaceutically acceptable carriers aresterile. Water is a preferred carrier when the Compounds of theInvention are administered intravenously. Saline solutions and aqueousdextrose and glycerol solutions can also be employed as liquid carriers,particularly for injectable solutions. Suitable pharmaceutical carriersalso include excipients such as starch, glucose, lactose, sucrose,gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerolmonostearate, talc, sodium chloride, dried skim milk, glycerol,propylene, glycol, water, ethanol and the like. The presentcompositions, if desired, can also contain minor amounts of wetting oremulsifying agents, or pH buffering agents.

The present compositions can take the form of solutions, suspensions,emulsion, tablets, pills, pellets, capsules, capsules containingliquids, powders, sustained-release formulations, suppositories,emulsions, aerosols, sprays, suspensions, or any other form suitable foruse. In one embodiment, the pharmaceutically acceptable carrier is acapsule (see e.g., U.S. Pat. No. 5,698,155). Other examples of suitablepharmaceutical carriers are described in “Remington's PharmaceuticalSciences” by E. W. Martin.

In a preferred embodiment, the Compounds of the Invention are formulatedin accordance with routine procedures as a pharmaceutical compositionadapted for intravenous administration to animals, particularly humanbeings. Typically, the carriers or vehicles for intravenousadministration are sterile isotonic aqueous buffer solutions. Wherenecessary, the compositions can also include a solubilizing agent.Compositions for intravenous administration can optionally comprise alocal anesthetic such as lignocaine to ease pain at the site of theinjection. Generally, the ingredients are supplied either separately ormixed together in unit dosage form, for example, as a dry lyophilizedpowder or water free concentrate in a hermetically sealed container suchas an ampoule or sachette indicating the quantity of active agent. Wherea Compound of the Invention is to be administered by infusion, it can bedispensed, for example, with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the Compound of theInvention is administered by injection, an ampoule of sterile water forinjection or saline can be provided so that the ingredients can be mixedprior to administration.

Compositions for oral delivery can be in the form of tablets, lozenges,aqueous or oily suspensions, granules, powders, emulsions, capsules,syrups, or elixirs, for example. Orally administered compositions cancontain one or more optionally agents, for example, sweetening agentssuch as fructose, aspartame or saccharin; flavoring agents such aspeppermint, oil of wintergreen, or cherry; coloring agents; andpreserving agents, to provide a pharmaceutically palatable preparation.Moreover, where in tablet or pill form, the compositions can be coatedto delay disintegration and absorption in the gastrointestinal tractthereby providing a sustained action over an extended period of time.Selectively permeable membranes surrounding an osmotically activedriving compound are also suitable for orally administered compounds. Inthese later platforms, fluid from the environment surrounding thecapsule is imbibed by the driving compound, which swells to displace theagent or agent composition through an aperture. These delivery platformscan provide an essentially zero order delivery profile as opposed to thespiked profiles of immediate release formulations. A time-delay materialsuch as glycerol monostearate or glycerol stearate can also be used.Oral compositions can include standard carriers such as mannitol,lactose, starch, magnesium stearate, sodium saccharine, cellulose,magnesium carbonate, etc. Such carriers are preferably of pharmaceuticalgrade.

The compositions can be intended for topical administration, in whichcase the carrier may be in the form of a solution, emulsion, ointment orgel base. The base, for example, can comprise one or more of thefollowing: petrolatum, lanolin, polyethylene glycols, beeswax, mineraloil, diluents such as water and alcohol, and emulsifiers andstabilizers. Thickening agents can be present in a composition fortopical administration. If intended for transdermal administration, thecomposition can be in the form of a transdermal patch or aniontophoresis device. Topical formulations can comprise a concentrationof a Compound of the Invention of from about 0.1% to about 10% w/v(weight per unit volume of composition).

The composition can be intended for rectal administration, in the form,e.g., of a suppository which will melt in the rectum and release theCompound of the Invention. The composition for rectal administration cancontain an oleaginous base as a suitable nonirritating excipient. Suchbases include, without limitation, lanolin, cocoa butter andpolyethylene glycol.

The composition can include various materials that modify the physicalform of a solid or liquid dosage unit. For example, the composition caninclude materials that form a coating shell around the activeingredients. The materials that form the coating shell are typicallyinert, and can be selected from, for example, sugar, shellac, and otherenteric coating agents. Alternatively, the active ingredients can beencased in a gelatin capsule.

The compositions can consist of gaseous dosage units, e.g., it can be inthe form of an aerosol. The term aerosol is used to denote a variety ofsystems ranging from those of colloidal nature to systems consisting ofpressurized packages. Delivery can be by a liquefied or compressed gasor by a suitable pump system that dispenses the active ingredients.Aerosols of Compounds of the Invention can be delivered in single phase,bi-phasic, or tri-phasic systems in order to deliver the Compound(s) ofthe Invention. Delivery of the aerosol includes the necessary container,activators, valves, subcontainers, Spacers and the like, which togethercan form a kit. Preferred aerosols can be determined by one skilled inthe art, without undue experimentation.

Whether in solid, liquid or gaseous form, the compositions of thepresent invention can comprise a pharmacological agent used in thetreatment of cancer, an autoimmune disease or an infectious disease.

The pharmaceutical compositions can be prepared using methodology wellknown in the pharmaceutical art. For example, a composition intended tobe administered by injection can be prepared by combining a Compound ofthe Invention with water so as to form a solution. A surfactant can beadded to facilitate the formation of a homogeneous solution orsuspension. Surfactants are compounds that non-covalently interact witha Compound of the Invention so as to facilitate dissolution orhomogeneous suspension of the active compound in the aqueous deliverysystem.

Therapeutic Uses of the Compounds of the Invention

The Compounds of the Invention are useful for treating cancer, anautoimmune disease or an infectious disease in an animal.

Treatment of Cancer

The Compounds of the Invention are useful for inhibiting themultiplication of a tumor cell or cancer cell, or for treating cancer inan animal. The Compounds of the Invention can be used accordingly in avariety of settings for the treatment of animal cancers. TheDrug-Linker-Ligand Conjugates can be used to deliver a Drug or Drug unitto a tumor cell or cancer cell. Without being bound by theory, in oneembodiment, the Ligand unit of a Compound of the Invention binds to orassociates with a cancer-cell or a tumor-cell-associated antigen, andthe Compound of the Invention can be taken up inside a tumor cell orcancer cell through receptor-mediated endocytosis. The antigen can beattached to a tumor cell or cancer cell or can be an extracellularmatrix protein associated with the tumor cell or cancer cell. Onceinside the cell, one or more specific peptide sequences within theLinker unit are hydrolytically cleaved by one or more tumor-cell orcancer-cell-associated proteases, resulting in release of a Drug or aDrug-Linker Compound. The released Drug or Drug-Linker Compound is thenfree to migrate in the cytosol and induce cytotoxic activities. In analternative embodiment, the Drug or Drug unit is cleaved from theCompound of the Invention outside the tumor cell or cancer cell, and theDrug or Drug-Linker Compound subsequently penetrates the cell.

In one embodiment, the Ligand unit binds to the tumor cell or cancercell.

In another embodiment, the Ligand unit binds to a tumor cell or cancercell antigen which is on the surface of the tumor cell or cancer cell.

In another embodiment, the Ligand unit binds to a tumor cell or cancercell antigen which is an extracellular matrix protein associated withthe tumor cell or cancer cell.

In one embodiment, the tumor cell or cancer cell is of the type of tumoror cancer that the animal needs treatment or prevention of.

The specificity of the Ligand unit for a particular tumor cell or cancercell can be important for determining those tumors or cancers that aremost effectively treated. For example, Compounds of the Invention havinga BR96 Ligand unit can be useful for treating antigen positivecarcinomas including those of the lung, breast, colon, ovaries, andpancreas. Compounds of the Invention having an Anti-CD30 or an anti-CD40Ligand unit can be useful for treating hematologic malignancies.

Other particular types of cancers that can be treated with Compounds ofthe Invention include, but are not limited to, those disclosed in Table3.

TABLE 3 Solid tumors, including but not limited to: fibrosarcomamyxosarcoma liposarcoma chondrosarcoma osteogenic sarcoma chordomaangiosarcoma endotheliosarcoma lymphangiosarcomalymphangioendotheliosarcoma synovioma mesothelioma Ewing's tumorleiomyosarcoma rhabdomyosarcoma colon cancer colorectal cancer kidneycancer pancreatic cancer bone cancer breast cancer ovarian cancerprostate cancer esophogeal cancer stomach cancer oral cancer nasalcancer throat cancer squamous cell carcinoma basal cell carcinomaadenocarcinoma sweat gland carcinoma sebaceous gland carcinoma papillarycarcinoma papillary adenocarcinomas cystadenocarcinoma medullarycarcinoma bronchogenic carcinoma renal cell carcinoma hepatoma bile ductcarcinoma choriocarcinoma seminoma embryonal carcinoma Wilms' tumorcervical cancer uterine cancer testicular cancer small cell lungcarcinoma bladder carcinoma lung cancer epithelial carcinoma gliomaglioblastoma multiforme astrocytoma medulloblastoma craniopharyngiomaependymoma pinealoma hemangioblastoma acoustic neuroma oligodendrogliomameningioma skin cancer melanoma neuroblastoma retinoblastoma blood-bornecancers, including but not limited to: acute lymphoblastic leukemia“ALL” acute lymphoblastic B-cell leukemia acute lymphoblastic T-cellleukemia acute myeloblastic leukemia “AML” acute promyelocytic leukemia“APL” acute monoblastic leukemia acute erythroleukemic leukemia acutemegakaryoblastic leukemia acute myelomonocytic leukemia acutenonlymphocyctic leukemia acute undifferentiated leukemia chronicmyelocytic leukemia “CML” chronic lymphocytic leukemia “CLL” hairy cellleukemia multiple myeloma acute and chronic leukemias: lymphoblasticmyelogenous lymphocytic myelocytic leukemias Lymphomas: Hodgkin'sdisease non-Hodgkin's Lymphoma Multiple myeloma Waldenström'smacroglobulinemia Heavy chain disease Polycythemia vera

The Compounds of the Invention can also be used as chemotherapeutics inthe untargeted form. For example, the Drugs themselves, or theDrug-Linker Compounds are useful for treating ovarian, CNS, renal, lung,colon, melanoma, or hematologic cancers or tumors.

The Compounds of the Invention provide Conjugation specific tumor orcancer targeting, thus reducing general toxicity of these compounds. TheLinker units stabilize the Compounds of the Invention in blood, yet arecleavable by tumor-specific proteases within the cell, liberating aDrug.

Multi-Modality Therapy for Cancer

Cancer, including, but not limited to, a tumor, metastasis, or anydisease or disorder characterized by uncontrolled cell growth, can betreated or prevented by administration of a Compound of the Invention.

In other embodiments, the invention provides methods for treating orpreventing cancer, comprising administering to an animal in need thereofan effective amount of a Compound of the Invention and achemotherapeutic agent. In one embodiment the chemotherapeutic agent isthat with which treatment of the cancer has not been found to berefractory. In another embodiment, the chemotherapeutic agent is thatwith which the treatment of cancer has been found to be refractory. TheCompounds of the Invention can be administered to an animal that hasalso undergone surgery as treatment for the cancer.

In one embodiment, the additional method of treatment is radiationtherapy.

In a specific embodiment, the Compound of the Invention is administeredconcurrently with the chemotherapeutic agent or with radiation therapy.In another specific embodiment, the chemotherapeutic agent or radiationtherapy is administered prior or subsequent to administration of aCompound of the Invention, preferably at least an hour, five hours, 12hours, a day, a week, a month, more preferably several months (e.g., upto three months), prior or subsequent to administration of a Compound ofthe Invention.

A chemotherapeutic agent can be administered over a series of sessions,any one or a combination of the chemotherapeutic agents listed in Table4 can be administered. With respect to radiation, any radiation therapyprotocol can be used depending upon the type of cancer to be treated.For example, but not by way of limitation, x-ray radiation can beadministered; in particular, high-energy megavoltage (radiation ofgreater that 1 MeV energy) can be used for deep tumors, and electronbeam and orthovoltage x-ray radiation can be used for skin cancers.Gamma-ray emitting radioisotopes, such as radioactive isotopes ofradium, cobalt and other elements, can also be administered.

Additionally, the invention provides methods of treatment of cancer witha Compound of the Invention as an alternative to chemotherapy orradiation therapy where the chemotherapy or the radiation therapy hasproven or can prove too toxic, e.g., results in unacceptable orunbearable side effects, for the subject being treated. The animal beingtreated can, optionally, be treated with another cancer treatment suchas surgery, radiation therapy or chemotherapy, depending on whichtreatment is found to be acceptable or bearable.

The Compounds of the Invention can also be used in an in vitro or exvivo fashion, such as for the treatment of certain cancers, including,but not limited to leukemias and lymphomas, such treatment involvingautologous stem cell transplants. This can involve a multi-step processin which the animal's autologous hematopoietic stem cells are harvestedand purged of all cancer cells, the patient's remaining bone-marrow cellpopulation is then eradicated via the administration of a high dose of aCompound of the Invention with or without accompanying high doseradiation therapy, and the stem cell graft is infused back into theanimal. Supportive care is then provided while bone marrow function isrestored and the animal recovers.

Multi-Drug Therapy for Cancer

The present invention includes methods for treating cancer, comprisingadministering to an animal in need thereof an effective amount of aCompound of the Invention and another therapeutic agent that is ananti-cancer agent. Suitable anticancer agents include, but are notlimited to, methotrexate, taxol, L-asparaginase, mercaptopurine,thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide,nitrosoureas, cisplatin, carboplatin, mitomycin, dacarbazine,procarbizine, topotecan, nitrogen mustards, cytoxan, etoposide,5-fluorouracil, BCNU, irinotecan, camptothecins, bleomycin, doxorubicin,idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone,asparaginase, vinblastine, vincristine, vinorelbine, paclitaxel, anddocetaxel. In a preferred embodiment, the anti-cancer agent includes,but is not limited to, a drug listed in Table 4.

TABLE 4 Alkylating agents Nitrogen mustards: cyclophosphamide Ifosfamidetrofosfamide Chlorambucil Nitrosoureas: carmustine (BCNU) Lomustine(CCNU) Alkylsulphonates busulfan Treosulfan Triazenes: DacarbazinePlatinum containing compounds: Cisplatin carboplatin Plant AlkaloidsVinca alkaloids: vincristine Vinblastine Vindesine Vinorelbine Taxoids:paclitaxel Docetaxol DNA Topoisomerase Inhibitors Epipodophyllins:etoposide Teniposide Topotecan 9-aminocamptothecin camptothecincrisnatol mitomycins: Mitomycin C Anti-metabolites Anti-folates: DHFRinhibitors: methotrexate Trimetrexate IMP dehydrogenase Inhibitors:mycophenolic acid Tiazofurin Ribavirin EICAR Ribonuclotide reductaseInhibitors: hydroxyurea deferoxamine Pyrimidine analogs: Uracil analogs5-Fluorouracil Floxuridine Doxifluridine Ratitrexed Cytosine analogscytarabine (ara C) Cytosine arabinoside fludarabine Purine analogs:mercaptopurine Thioguanine Hormonal therapies: Receptor antagonists:Anti-estrogen Tamoxifen Raloxifene megestrol LHRH agonists: goscrclinLeuprolide acetate Anti-androgens: flutamide bicalutamideRetinoids/Deltoids Vitamin D3 analogs: EB 1089 CB 1093 KH 1060Photodynamic therapies: vertoporfin (BPD-MA) Phthalocyaninephotosensitizer Pc4 Demethoxy-hypocrellin A (2BA-2-DMHA) Cytokines:Interferon-α Interferon-γ Tumor necrosis factor Others: Isoprenylationinhibitors: Lovastatin Dopaminergic neurotoxins:1-methyl-4-phenylpyridinium ion Cell cycle inhibitors: staurosporineActinomycins: Actinomycin D Dactinomycin Bleomycins: bleomycin A2Bleomycin B2 Peplomycin Anthracyclines: daunorubicin Doxorubicin(adriamycin) Idarubicin Epirubicin Pirarubicin Zorubicin MitoxantroneMDR inhibitors: verapamil Ca²⁺ATPase inhibitors: thapsigargin

Treatment of Autoimmune Diseases

The Compounds of the Invention are useful for killing or inhibiting thereplication of a cell that produces an autoimmune disease or fortreating an autoimmune disease. The Compounds of the Invention can beused accordingly in a variety of settings for the treatment of anautoimmune disease in an animal. The Drug-Linker-Ligand Conjugates canbe used to deliver a Drug to a target cell. Without being bound bytheory, in one embodiment, the Drug-Linker-Ligand Conjugate associateswith an antigen on the surface of a target cell, and the Compound of theInvention is then taken up inside a target-cell throughreceptor-mediated endocytosis. Once inside the cell, one or morespecific peptide sequences within the Linker unit are enzymatically orhydrolytically cleaved, resulting in release of a Drug. The releasedDrug is then free to migrate in the cytosol and induce cytotoxicactivities. In an alternative embodiment, the Drug is cleaved from theCompound of the Invention outside the target cell, and the Drugsubsequently penetrates the cell.

In one embodiment, the Ligand unit binds to an autoimmune antigen.

In another embodiment, the Ligand unit binds to an autoimmune antigenwhich is on the surface of a cell.

In another embodiment, the target cell is of the type of cell thatproduces the autoimmune antigen which causes the disease the animalneeds treatment or prevention of.

In a preferred embodiment, the Ligand binds to activated lympocytes thatare associated with the autoimmune diesease state.

In a further embodiment, the Compounds of the Invention kill or inhibitthe multiplication of cells that produce an auto-immune antibodyassociated with a particular autoimmune disease.

Particular types of autoimmune diseases that can be treated with theCompounds of the Invention include, but are not limited to,Th2-lymphocyte related disorders (e.g., atopic dermatitis, atopicasthma, rhinoconjunctivitis, allergic rhinitis, Omenn's syndrome,systemic sclerosis, and graft versus host disease); Th1lymphocyte-related disorders (e.g., rheumatoid arthritis, multiplesclerosis, psoriasis, Sjorgren's syndrome, Hashimoto's thyroiditis,Grave's disease, primary biliary cirrhosis, Wegener's granulomatosis,and tuberculosis); activated B lymphocyte-related disorders (e.g.,systemic lupus erythematosus, Goodpasture's syndrome, rheumatoidarthritis, and type I diabetes); and those disclosed in Table 5.

TABLE 5 Active Chronic Hepatitis Addison's Disease Allergic AlveolitisAllergic Reaction Allergic Rhinitis Alport's Syndrome AnaphlaxisAnkylosing Spondylitis Anti-phosholipid Syndrome Arthritis AscariasisAspergillosis Atopic Allergy Atropic Dermatitis Atropic RhinitisBehcet's Disease Bird-Fancier's Lung Bronchial Asthma Caplan's SyndromeCardiomyopathy Celiac Disease Chagas' Disease Chronic GlomerulonephritisCogan's Syndrome Cold Agglutinin Disease Congenital Rubella InfectionCREST Syndrome Crohn's Disease Cryoglobulinemia Cushing's SyndromeDermatomyositis Discoid Lupus Dressler's Syndrome Eaton-Lambert SyndromeEchovirus Infection Encephalomyelitis Endocrine opthalmopathyEpstein-Barr Virus Infection Equine Heaves Erythematosis Evan's SyndromeFelty's Syndrome Fibromyalgia Fuch's Cyclitis Gastric AtrophyGastrointestinal Allergy Giant Cell Arteritis GlomerulonephritisGoodpasture's Syndrome Graft v. Host Disease Graves' DiseaseGuillain-Barre Disease Hashimoto's Thyroiditis Hemolytic AnemiaHenoch-Schonlein Purpura Idiopathic Adrenal Atrophy Idiopathic PulmonaryFibritis IgA Nephropathy Inflammatory Bowel Diseases Insulin-dependentDiabetes Mellitus Juvenile Arthritis Juvenile Diabetes Mellitus (Type I)Lambert-Eaton Syndrome Laminitis Lichen Planus Lupoid Hepatitis LupusLymphopenia Meniere's Disease Mixed Connective Tissue Disease MultipleSclerosis Myasthenia Gravis Pernicious Anemia Polyglandular SyndromesPresenile Dementia Primary Agammaglobulinemia Primary Biliary CirrhosisPsoriasis Psoriatic Arthritis Raynauds Phenomenon Recurrent AbortionReiter's Syndrome Rheumatic Fever Rheumatoid Arthritis Sampter'sSyndrome Schistosomiasis Schmidt's Syndrome Scleroderma Shulman'sSyndrome Sjorgen's Syndrome Stiff-Man Syndrome Sympathetic OphthalmiaSystemic Lupus Erythematosis Takayasu's Arteritis Temporal ArteritisThyroiditis Thrombocytopenia Thyrotoxicosis Toxic Epidermal NecrolysisType B Insulin Resistance Type I Diabetes Mellitus Ulcerative ColitisUveitis Vitiligo Waldenstrom's Macroglobulemia Wegener's Granulomatosis

Multi-Drug Therapy of Autoimmune Diseases

The present invention also provides methods for treating an autoimmunedisease, comprising administering to an animal in need thereof aneffective amount of a Compound of the Invention and another therapeuticagent that known for the treatment of an autoimmune disease. In oneembodiment, the anti-autoimmune disease agent includes, but is notlimited to, agents listed in Table 6.

TABLE 6 cyclosporine cyclosporine A mycophenylate mofetil sirolimustacrolimus enanercept prednisone azathioprine methotrexatecyclophosphamide prednisone aminocaproic acid chloroquinehydroxychloroquine hydrocortisone dexamethasone chlorambucil DHEAdanazol bromocriptine meloxicam infliximab

Treatment of Infectious Diseases

The Compounds of the Invention are useful for killing or inhibiting themultiplication of a cell that produces an infectious disease or fortreating an infectious disease. The Compounds of the Invention can beused accordingly in a variety of settings for the treatment of aninfectious disease in an animal. The Drug-Linker-Ligand Conjugates canbe used to deliver a Drug to a target cell. Without being bound bytheory, in one embodiment, the Drug-Linker-Ligand Conjugate associateswith an antigen on the surface of a target cell, and the Compound of theInvention is then taken up inside a target-cell throughreceptor-mediated endocytosis. Once inside the cell, one or morespecific peptide sequences within the Linker unit are enzymatically orhydrolytically cleaved, resulting in release of a Drug. The releasedDrug is then free to migrate in the cytosol and induce cytotoxicactivities. In an alternative embodiment, the Drug is cleaved from theCompound of the Invention outside the target cell, and the Drugsubsequently penetrates the cell.

In one embodiment, the Ligand unit binds to the infectious disease cell.

In one embodiment, the infectious disease type of infectious diseasethat the animal needs treatment or prevention of.

In one embodiment, the Compounds of the Invention kill or inhibit themultiplication of cells that produce a particular infectious disease.

Particular types of infectious diseases that can be treated with theCompounds of the Invention include, but are not limited to, thosedisclosed in Table 7.

TABLE 7 Bacterial Diseases: Diptheria Pertussis Occult BacteremiaUrinary Tract Infection Gastroenteritis Cellulitis EpiglottitisTracheitis Adenoid Hypertrophy Retropharyngeal Abcess Impetigo EcthymaPneumonia Endocarditis Septic Arthritis Pneumococcal PeritonitisBactermia Meningitis Acute Purulent Meningitis Urethritis CervicitisProctitis Pharyngitis Salpingitis Epididymitis Gonorrhea SyphilisListeriosis Anthrax Nocardiosis Salmonella Typhoid Fever DysenteryConjuntivitis Sinusitis Brucellosis Tullaremia Cholera Bubonic PlagueTetanus Necrotizing Enteritis Actinomycosis Mixed Anaerobic InfectionsSyphilis Relapsing Fever Leptospirosis Lyme Disease Rat Bite FeverTuberculosis Lymphadenitis Leprosy Chlamydia Chlamydial PneumoniaTrachoma Inclusion Conjunctivitis Systemic Fungal Diseases:Histoplamosis Coccicidiodomycosis Blastomycosis SporotrichosisCryptococcsis Systemic Candidiasis Aspergillosis Mucormycosis MycetomaChromomycosis Rickettsial Diseases: Typhus Rocky Mountain Spotted FeverEhrlichiosis Eastern Tick-Borne Rickettsioses Rickettsialpox Q FeverBartonellosis Parasitic Diseases: Malaria Babesiosis African SleepingSickness Chagas' Disease Leishmaniasis Dum-Dum Fever ToxoplasmosisMeningoencephalitis Keratitis Entamebiasis Giardiasis CryptosporidiasisIsosporiasis Cyclosporiasis Microsporidiosis Ascariasis WhipwormInfection Hookworm Infection Threadworm Infection Ocular Larva MigransTrichinosis Guinea Worm Disease Lymphatic Filariasis Loiasis RiverBlindness Canine Heartworm Infection Schistosomiasis Swimmer's ItchOriental Lung Fluke Oriental Liver Fluke Fascioliasis FasciolopsiasisOpisthorchiasis Tapeworm Infections Hydatid Disease Alveolar HydatidDisease Viral Diseases: Measles Subacute sclerosing panencephalitisCommon Cold Mumps Rubella Roseola Fifth Disease Chickenpox Respiratorysyncytial virus infection Croup Bronchiolitis Infectious MononucleosisPoliomyelitis Herpangina Hand-Foot-and-Mouth Disease Bornholm DiseaseGenital Herpes Genital Warts Aseptic Meningitis Myocarditis PericarditisGastroenteritis Acquired Immunodeficiency Syndrome (AIDS) Reye'sSyndrome Kawasaki Syndrome Influenza Bronchitis Viral “Walking”Pneumonia Acute Febrile Respiratory Disease Acute pharyngoconjunctivalfever Epidemic keratoconjunctivitis Herpes Simplex Virus 1 (HSV-1)Herpes Simples Virus 2 (HSV-2) Shingles Cytomegalic Inclusion DiseaseRabies Progressive Multifocal Leukoencephalopathy Kuru Fatal FamilialInsomnia Creutzfeldt-Jakob Disease Gerstmann-Straussler-ScheinkerDisease Tropical Spastic Paraparesis Western Equine EncephalitisCalifornia Encephalitis St. Louis Encephalitis Yellow Fever DengueLymphocytic choriomeningitis Lassa Fever Hemorrhagic Fever HantvirusPulmonary Syndrome Marburg Virus Infections Ebola Virus InfectionsSmallpox

Multi-Drug Therapy of Infectious Diseases

The present invention also provides methods for treating an infectiousdisease, comprising administering to an animal in need thereof aCompound of the Invention and another therapeutic agent that is ananti-infectious disease agent. In one embodiment, the anti-infectiousdisease agent is, but not limited to, agents listed in Table 8.

TABLE 8 Antibacterial Agents: β-Lactam Antibiotics: Penicillin GPenicillin V Cloxacilliin Dicloxacillin Methicillin Nafcillin OxacillinAmpicillin Amoxicillin Bacampicillin Azlocillin CarbenicillinMezlocillin Piperacillin Ticarcillin Aminoglycosides: AmikacinGentamicin Kanamycin Neomycin Netilmicin Streptomycin TobramycinMacrolides: Azithromycin Clarithromycin Erythromycin LincomycinClindamycin Tetracyclines: Demeclocycline Doxycycline MinocyclineOxytetracycline Tetracycline Quinolones: Cinoxacin Nalidixic AcidFluoroquinolones: Ciprofloxacin Enoxacin Grepafloxacin LevofloxacinLomefloxacin Norfloxacin Ofloxacin Sparfloxacin TrovafloxicinPolypeptides: Bacitracin Colistin Polymyxin B Sulfonamides:Sulfisoxazole Sulfamethoxazole Sulfadiazine Sulfamethizole SulfacetamideMiscellaneous Antibacterial Agents: Trimethoprim SulfamethazoleChloramphenicol Vancomycin Metronidazole Quinupristin DalfopristinRifampin Spectinomycin Nitrofurantoin Antiviral Agents: GeneralAntiviral Agents: Idoxuradine Vidarabine Trifluridine AcyclovirFamicyclovir Pencicyclovir Valacyclovir Gancicyclovir FoscarnetRibavirin Amantadine Rimantadine Cidofovir Antisense OligonucleotidesImmunoglobulins Inteferons Drugs for HIV infection: ZidovudineDidanosine Zalcitabine Stavudine Lamivudine Nevirapine DelavirdineSaquinavir Ritonavir Indinavir Nelfinavir

Other Therapeutic Agents

The present methods can further comprise the administration of aCompound of the Invention and an additional therapeutic agent orpharmaceutically acceptable salts or solvates thereof. The Compound ofthe Invention and the other therapeutic agent can act additively or,more preferably, synergistically. In a preferred embodiment, acomposition comprising a Compound of the Invention is administeredconcurrently with the administration of one or more additionaltherapeutic agent(s), which can be part of the same composition or in adifferent composition from that comprising the Compound of theInvention. In another embodiment, a Compound of the Invention isadministered prior to or subsequent to administration of anothertherapeutic agent(s).

In the present methods for treating cancer, an autoimmune disease or aninfectious disease, the other therapeutic agent can be an antiemeticagent. Suitable antiemetic agents include, but are not limited to,metoclopromide, domperidone, prochlorperazine, promethazine,chlorpromazine, trimethobenzamide, ondansetron, granisetron,hydroxyzine, acethylleucine monoethanolamine, alizapride, azasetron,benzquinamide, bietanautine, bromopride, buclizine, clebopride,cyclizine, dimenhydrinate, diphenidol, dolasetron, meclizine,methallatal, metopimazine, nabilone, oxyperndyl, pipamazine,scopolamine, sulpiride, tetrahydrocannabinols, thiethylperazine,thioproperazine and tropisetron.

In another embodiment, the other therapeutic agent can be anhematopoietic colony stimulating factor. Suitable hematopoietic colonystimulating factors include, but are not limited to, filgrastim,sargramostim, molgramostim and erythropoietin alfa.

In still another embodiment, the other therapeutic agent can be anopioid or non-opioid analgesic agent. Suitable opioid analgesic agentsinclude, but are not limited to, morphine, heroin, hydromorphone,hydrocodone, oxymorphone, oxycodone, metopon, apomorphine, normorphine,etorphine, buprenorphine, meperidine, lopermide, anileridine,ethoheptazine, piminidine, betaprodine, diphenoxylate, fentanil,sufentanil, alfentanil, remifentanil, levorphanol, dextromethorphan,phenazocine, pentazocine, cyclazocine, methadone, isomethadone andpropoxyphene. Suitable non-opioid analgesic agents include, but are notlimited to, aspirin, celecoxib, rofecoxib, diclofinac, diflusinal,etodolac, fenoprofen, flurbiprofen, ibuprofen, ketoprofen, indomethacin,ketorolac, meclofenamate, mefanamic acid, nabumetone, naproxen,piroxicam and sulindac.

The following examples are provided by way of illustration and notlimitation.

Examples

Materials and Methods. Commercially available reagents and solvents wereobtained as follows: HPLC-grade solvents, Fisher Scientific (Atlanta,Ga.); anhydrous solvents, Aldrich (St. Louis, Mo.);diisopropylazodicarboxylate (DIAD, 95%), Lancaster (Lancashire,England); 4-aminobenzyl alcohol, Alfa Aesar (Ward Hill, Mass.);L-citrulline, Novabiochem (Laufelfingen, Switzerland); all other aminoacids, Advanced ChemTech (Louisville, Ky.) or Novabiochem (Laufelfingen,Switzerland); (1S,2R)-(+)-norephedrine and other commercially availablereagents, Aldrich or Acros; all coupling reagents were acquired fromNovabiochem or Aldrich. All solvents used as reaction media are assumedto be anhydrous unless otherwise indicated. 1H-NMR spectra were recordedon either a Varian Gemini at 300 MHz or Varian Mercury 400 MHzspectrophotometer. Flash column chromatography was performed using230-400 mesh ASTM silica gel from Fisher. Analtech silica gel GHLFplates were used for thin-layer chromatography. Analytical HPLC wasperformed using a Waters Alliance system using a photodiode arraydetector. Preparative HPLC purification was performed using a VarianProstar system that had either a photodiode array or dual wavelengthdetector. Combustion analyses were determined by QuantitativeTechnologies, Inc., Whitehouse, N.J.

Examples 5-12 relate to Drugs that can be used as Drug units in theinvention.

Example 1 Preparation of Compound 21

Fmoc-(L)-val-(L)-cit-PAB-OH (19) (14.61 g, 24.3 mmol, 1.0 eq., U.S. Pat.No. 6,214,345 to Firestone et al.) was diluted with DMF (120 mL, 0.2 M)and to this solution was added a diethylamine (60 mL). The reaction wasmonitored by HPLC and found to be complete in 2 h. The reaction mixturewas concentrated and the resulting residue was precipitated using ethylacetate (about 100 mL) under sonication over for 10 min. Ether (200 mL)was added and the precipitate was further sonicated for 5 min. Thesolution was allowed to stand for 30 min. without stirring and was thenfiltered and dried under high vacuum to provide Val-cit-PAB-OH, whichwas used in the next step without further purification. Yield: 8.84 g(96%). Val-cit-PAB-OH (8.0 g, 21 mmol) was diluted with DMF (110 mL) andthe resulting solution was treated with MC—OSu (Willner et al.,Bioconjugate Chem. 4, 521, 1993, 6.5 g, 21 mmol, 1.0 eq.). Reaction wascomplete according to HPLC after 2 h. The reaction mixture wasconcentrated and the resulting oil was precipitated using ethyl acetate(50 mL). After sonicating for 15 min, ether (400 mL) was added and themixture was sonicated further until all large particles were broken up.The solution was then filtered and the solid dried to provide Compound20 as an off-white solid. Yield: 11.63 g (96%); ES-MS m/z 757.9 [M−H]−

Compound 20 (8.0 g, 14.0 mmol) was diluted with DMF (120 mL, 0.12 M) andto the resulting solution was added bis(4-nitrophenyl)carbonate (8.5 g,28.0 mmol, 2.0 eq.) and diisopropylethylamine (3.66 mL, 21.0 mmol, 1.5eq.). The reaction was complete in 1 h according to HPLC. The reactionmixture was concentrated to provide an oil that was precipitated withEtOAc, and then triturated using EtOAc (about 25 mL). The solute wasfurther precipitated with ether (about 200 mL) and triturated for 15min. The solid was filtered and dried under high vacuum to provideCompound 21 which was 93% pure according to HPLC and used in the nextstep without further purification. Yield: 9.7 g (94%).

Example 2 Preparation of Compound 27

Compound 26 (2.0 g, 2.31 mmol, 1.0 eq.) was diluted with dichloromethane(30 mL), and to the resulting solution was addedbis(4-nitrophenyl)carbonate (2.72 g, 8.94 mmol, 3.8 eq.) followed bydiisopropylethylamine (1.04 mL, 5.97 mmol, 2.6 eq.). The reaction wascomplete in 3 d, according to HPLC. The reaction mixture wasconcentrated and the resulting residue was triturated using ether, thenfiltered and dried under high vacuum to provide Compound 27 as a yellowsolid (2.37 g, 97%).

Example 3 Preparation of Compound 28

Fmoc-phe-lys(Mtr)-OH (24) (0.5 g, 0.63 mmol, U.S. Pat. No. 6,214,345 toFirestone et al.) was diluted with dichloromethane to a concentration of0.5 M and to this solution was added diethylamine in an amount that wasapproximately one-third of the volume of the Compound 24/dichloromethanesolution. The reaction was allowed to stir and was monitored using HPLC.It was shown to be complete by HPLC in 3 h. The reaction mixture wasconcentrated in vacuo, and the resulting residue was diluted with ethylacetate and then reconcentrated. The resulting residue was trituratedusing ether and filtered. The residual solid was diluted withdichloromethane to a concentration of 0.2M, and to the resultingsolution was added MC—OSu (0.20 g, 0.63 mmol, 1.0 eq.) anddiisopropylethylamine (0.12 mL, 0.70 mmol, 1.1 eq.). The reactionmixture was allowed to stir under a nitrogen atmosphere for 16 h, afterwhich time HPLC showed very little starting material. The reactionmixture was then concentrated and the resulting residue was trituratedusing ether to provide Compound 28 as a colored solid. Yield: 100 mg(21%); ES-MS m/z 757.9 M−H⁻.

Example 4 Preparation of Compound 19A

Compound 19 (1.0 g, 1.66 mmol) was diluted with DMF (10 mL) and to theresulting solution was added bis(4-nitrophenyl)carbonate (1.0 g, 3.3mmol, 2.0 eq.).

The reaction mixture was immediately treated with diisopropylethylamine(0.43 mL, 2.5 mmol, 1.5 eq.) and the reaction was allowed to stir underan argon atmosphere. The reaction was complete in 2.5 h according toHPLC. The reaction mixture was concentrated to provide a light brown oilthat was precipitated using ethyl acetate (5 mL), then precipitatedagain using ether (about 100 mL). The resulting precipitate was allowedto stand for 30 min, and was then filtered and dried under high vacuumto provide Compound 19a as an off-white powder. Yield: 1.05 g (83%);ES-MS m/z 767.2 [M+H⁺; UV λ_(max) 215, 256 nm.

Example 5 Preparation of Compound 49

Compound 49 was made according to General Procedure D usingFmoc-Me-val-val-dil-O-t-Bu 39 (0.40 g, 0.57 mmol) as the tripeptide andBoc-dap-nor 44 (0.26 g, 0.62 mmol, 1.1 eq.) as the dipeptide. Thereaction mixture was purified using flash column chromatography (silicagel column, eluant—100% EtOAc). Two Fmoc-containing products eluted: theFmoc derivative of Compound 49 (R_(f) 0.17 in 100% EtOAc) and what wasbelieved to be the Fmoc derivative of the TFA acetate of Compound 49(R_(f) 0.37). The products were combined to provide a white foam thatwas subjected to General Procedure E. Reaction was complete after 2 h.Solvents were removed to provide an oil that was purified using flashcolumn chromatography (eluant—9:1 Dichloromethane-methanol) to provideCompound 49.

Example 6 Preparation of Compound 50

Compound 50 was prepared by reacting tripeptide 42 and dipeptide 48according to General Procedure D using triethylamine (5.0 eq.) as thebase. After concentration of the reaction mixture, the resulting residuewas directly injected onto a reverse phase preparative-HPLC column(Varian Dynamax column 21.4 mm×25 cm, 5μ, 100 Å, using a gradient run ofMeCN and 0.1M TEA/CO₂ at 20 mL/min from 10% to 100% over 40 min followedby 100% MeCN for 20 min). The relevant fractions were pooled andconcentrated, and the resulting residue was diluted with 10 mL ofdichloromethane-ether (1:1). The solution was cooled to 0° C. and 1.0Methereal HCl was added dropwise (approx. 10 eq.). The precipitate,Compound 50, was filtered and dried and was substantially pure by HPLC.Yield: 71 mg (43%); ES-MS m/z 731.6 [M+H]⁺; UV λ_(max) 215, 238, 290 nm.Anal. Calc. C₄₀H₇₀N₆O₆.4H₂O.2HCl: C, 54.84; H, 9.20; N, 9.59. Found: C,55.12; H, 9.41; N, 9.82.

Example 7 Preparation of Compound 51

Compound 51 was prepared by reacting Fmoc-tripeptide 41 and dipeptide 46according to General Procedure D using triethylamine as the base. Afterconcentration of the reaction mixture, the residue was directly injectedonto a reverse phase preparative-HPLC column (Varian Dynamax column 21.4mm×25 cm, 5μ, 100 Å, using a gradient run of MeCN and 0.1M TEA/CO₂ at 20mL/min from 10% to 100% over 40 min followed by 100% MeCN for 20 min).The relevant fractions were pooled and concentrated to provide a whitesolid intermediate that was used in the next step without furtherpurification. ES-MS m/z 882.9 [M+NH₄]⁺, 899.9 [M+Na]⁺; UV λ_(max) 215,256 nm.

Deprotection of the white solid intermediate was performed according toGeneral Procedure E. The crude product was purified usingpreparative-HPLC (Varian Dynamax column 21.4 mm×25 cm, 5μ, 100 Å, usinga gradient run of MeCN and 0.1M TEA/CO₂ at 20 mL/min from 10% to 100%over 40 min followed by 100% MeCN for 20 min). The relevant fractionswere pooled and concentrated to provide Compound 51 as a sticky solid.ES-MS m/z 660.1 [M+H]⁺, 682.5 [M+Na]⁻; UV λ_(max) 215 nm.

Example 8 Preparation of Compound 52

Boc-dolaproine (0.33 g, 1.14 mmol) and(1S,2S)-(−)-1,2-diphenylethylenediamine (0.5 g, 2.28 mmol, 2.0 eq.) werediluted with dichloromethane, (10 mL) and to the resulting solution wasadded triethylamine (0.32 mL, 2.28 mmol, 2.0 eq.), then DEPC (0.39 mL,2.28 mmol, 2.0 eq.). After 4 h, additional DEPC (0.39 mL) was added andthe reaction was allowed to stir overnight. The reaction mixture wasconcentrated and the resulting residue was purified usingpreparative-HPLC (Varian Dynamax C18 column 21.4 mm×25 cm, 5μ, 100 Å,using a gradient run of MeCN and water at 20 mL/min from 10% to 100%over 40 min followed by 100% MeCN for 20 min). The relevant fractionswere pooled and concentrated to provide a yellow gummy solid peptideintermediate that was used without further purification. R_(f) 0.15(100% EtOAc); ES-MS m/z 482.4 [M+H⁺; UV λ_(max) 215, 256 nm.

The yellow gummy peptide intermediate (0.24 g, 0.50 mmol) was dilutedwith dichloromethane, and to the resulting solution was addeddiisopropylethylamine (0.18 mL, 1.0 mmol, 2.0 eq.) and Fmoc-Cl (0.15 g,0.55 mmol, 1.1 eq.). The reaction was allowed to stir for 3 h, afterwhich time HPLC showed a complete reaction. The reaction mixture wasconcentrated to an oil, and the oil was diluted with EtOAc and extractedsuccessively with 10% aqueous citric acid, water, saturated aqueoussodium bicarbonate, and brine. The EtOAc layer was dried, filtered, andconcentrated, and the resulting residue was purified using flash columnchromatography (silica gel 230-400 mesh; eluant gradient 4:1hexanes-EtOAc to 1:1 hexanes-EtOAc) to provide Compound 45 as a whitesolid. Yield: 0.37 g (46% overall); R_(f) 0.47 (1:1 hexanes-EtOAc);ES-MS m/z 704.5 [M+H]⁺, 721.4 [M+NH₄]⁺; UV λ_(max) 215, 256 nm.

Compound 52 was prepared by reacting tripeptide 42 (94 mg, 0.13 mmol)and dipeptide compound 45 (65 mg, 0.13 mmol) according to GeneralProcedure D (using 3.6 eq. of diisopropylethylamine as the base). Afterconcentration of the reaction mixture, the resulting residue was dilutedwith EtOAc and washed successively with 10% aqueous citric acid, water,saturated aqueous sodium bicarbonate, and brine. The organic phase wasdried, filtered and concentrated to provide a white solid residue whichwas diluted with dichloromethane and deprotected according to GeneralProcedure E. According to HPLC, reaction was complete after 2 h. Thereaction mixture was concentrated to an oil. The oil was diluted withDMSO, and the resulting solution was purified using a reverse phasepreparative-HPLC (Varian Dynamax column 21.4 mm×25 cm, 5μ, 100 Å, usinga gradient run of MeCN and 0.1% TFA at 20 mL/min from 10% to 100% over40 min followed by 100% MeCN for 20 min). Two products having similar UVspectra were isolated. The major product, Compound 52, was provided asan off-white solid. Overall yield: 24 mg (23%); ES-MS m/z 793.5 [M+H]⁺;UV λ_(max) 215 nm.

Example 9 Preparation of Compound 53

Boc-phenylalanine (1.0 g, 3.8 mmol) was added to a suspension of1,4-diaminobenzene.HCl (3.5 g, 19.0 mmol, 5.0 eq.) in triethylamine(10.7 mL, 76.0 mmol, 20 eq.) and dichloromethane (50 mL). To theresulting solution was added DEPC (3.2 mL, 19.0 mmol, 5.0 eq.) viasyringe. HPLC showed no remaining Boc-phe after 24 h. The reactionmixture was filtered, and the filtrate was concentrated to provide adark solid. The dark solid residue was partitioned between 1:1EtOAc-water, and the EtOAc layer was washed sequentially with water andbrine. The EtOAc layer was dried and concentrated to provide a darkbrown/red residue that was purified using HPLC (Varian Dynamax column41.4 mm×25 cm, 5μ, 100 Å, using a gradient run of MeCN and water at 45mL/min form 10% to 100% over 40 min followed by 100% MeCN for 20 min).The relevant fractions were combined and concentrated to provide ared-tan solid intermediate. Yield: 1.4 g (100%); ES-MS m/z 355.9 [M+H]⁻;UV λ_(max) 215, 265 nm; ¹H NMR (CDCl₃) δ 7.48 (1H, br s), 7.22-7.37 (5H,m), 7.12 (2H, d, J=8.7Hz), 7.61 (2H, d, J=8.7 Hz), 5.19 (1H, br s),4.39-4.48 (1H, m), 3.49 (2H, s), 3.13 (2H, d, J=5.7 Hz), 1.43 (9H, s).

The red-tan solid intermediate (0.5 g, 1.41 mmol) anddiisopropylethylamine (0.37 mL, 2.11 mmol, 1.5 eq.) were diluted withdichloromethane (10 mL), and to the resulting solution was added Fmoc-Cl(0.38 g, 1.41 mmol). The reaction was allowed to stir, and a white solidprecipitate formed after a few minutes. Reaction was complete accordingto HPLC after 1 h. The reaction mixture was filtered, and the filtratewas concentrated to provide an oil. The oil was precipitated with EtOAc,resulting in a reddish-white intermediate product, which was collectedby filtration and dried under vacuum. Yield: 0.75 g (93%); ES-MS m/z578.1 [M+H]⁺, 595.6 [M+NH₄]⁺.

The reddish-white intermediate (0.49 g, 0.85 mmol), was diluted with 10mL of dichloromethane, and then treated with 5 mL of trifluoroaceticacid. Reaction was complete in 30 min according to reverse-phase HPLC.The reaction mixture was concentrated and the resulting residue wasprecipitated with ether to provide an off-white solid. The off-whitesolid was filtered and dried to provide an amorphous powder, which wasadded to a solution of Boc-dap (0.24 g, 0.85 mmol) in dichloromethane(10 mL). To this solution was added triethylamine (0.36 mL, 2.5 mmol,3.0 eq.) and PyBrop (0.59 g, 1.3 mmol, 1.5 eq.). The reaction mixturewas monitored using reverse-phase HPLC. Upon completion, the reactionmixture was concentrated, and the resulting residue was diluted withEtOAc, and sequentially washed with 10% aqueous citric acid, water,saturated aqueous sodium bicarbonate, water, and brine. The EtOAc layerwas dried (MgSO₄), filtered, and concentrated. The resulting residue waspurified using flash column chromatography (silica gel) to provideCompound 47 as an off-white powder. Yield: 0.57 g (88%); ES-MS m/z 764.7[M+NH₄]⁺; UV λ_(max) 215, 265 nm; ¹H NMR (DMSO-d₆) δ (10.0-10.15 (1H,m), 9.63 (1H, br s), 8.42 (½H, d, J=8.4 Hz), 8.22 (1/2H, d, J=8.4 Hz),7.89 (2H, d, J=7.2 Hz), 7.73 (2H, d, J=7.6 Hz), 7.11-7.55 (13H, m),4.69-4.75 (1H, m) 4.46 (2H, d, J=6.8 Hz), 4.29 (1H, t, J=6.4 Hz), 3.29(3H, s), 2.77-3.47 (7H, m), 2.48-2.50 (3H, m), 2.25 (⅔H, dd, J=9.6, 7.2Hz), 1.41-1.96 (4H, m), 1.36 (9H, s), 1.07 (1H, d, J=6.4 Hz, rotationalisomer), 1.00 (1H, d, J=6.4 Hz, rotational isomer).

Tripeptide compound 42 (55 mg, 0.11 mmol) and dipeptide compound 47 (85mg, 0.11 mmol) were reacted according to General Procedure D (using 3.0eq. of diisopropylethylamine). After concentration of the reactionmixture, the resulting residue was diluted with EtOAc, and washedsequentially with 10% aqueous citric acid, water, saturated aqueoussodium bicarbonate, and brine. The EtOAc layer was dried, filtered andconcentrated to provide a yellow oil. The yellow oil was diluted withdichloromethane (10 mL) and deprotected according to General ProcedureE. According to HPLC, reaction was complete after 2 h. The reactionmixture was concentrated to provide an oil. The oil was diluted withDMSO, and the DMSO solution was purified using reverse phasepreparative-HPLC (Varian Dynamax column 21.4 mm×25 cm, 5μ, 100 Å, usinga gradient run of MeCN and 0.1% TFA at 20 mL/min from 10% to 100% over40 min followed by 100% MeCN for 20 min). The relevant fractions werecombined and concentrated to provide Compound 53 as an off-white solid.Overall yield: 42 mg (44% overall); ES-MS m/z 837.8 [M+H]⁺, 858.5[M+Na]⁺; UV λ_(max) 215, 248 nm.

Example 10 Preparation of Compound 54

Compound 54 was prepared according to K. Miyazaki, et al. Chem. Pharm.Bull. 1995, 43(10), 1706-18.

Example 11 Preparation of Compound 55

Compound 55 was synthesized in the same manner as Compound 54, but bysubstituting FmocMeVal-Ile-Dil-tBu (40) for FmocMeVal-Val-Dil-tBu (39)as the starting material.

Example 12 Preparation of Compound 56

Carbamic acid [(1S)-1-(azidomethyl)-2-phenylethyl]-1,1-dimethylethylester (0.56 g, 2 mmol, prepared as described in J. Chem. Research (S),1992, 391), was diluted with a 4 M solution of HCl in dioxane (10 mL)and the resulting solution allowed to stir for 2 hr at room temperature.Toluene (10 mL) was then added to the reaction, the reaction mixture wasconcentrated and the resulting residue was azeotropically dried undervacuum using toluene (3×15 mL), to provide a white solid intermediate.ES-MS m/z 177.1 [M+H]⁺.

The white solid intermediate was diluted with dichloromethane (5 mL) andto the resulting solution was added sequentially N-Boc-Dolaproine (0.58g, 1 eq.), triethylamine (780 μL, 3 eq.) and DEPC (406 μL, 1.2 eq.), andthe reaction mixture was allowed to stir for 2 h at room temperature.Reaction progress was monitored using reverse-phase HPLC. Uponcompletion of reaction as determined by HPLC, the reaction mixture wasdiluted with dichloromethane (30 mL), the dichloromethane layer waswashed successively with 10% aqueous citric acid (20 mL), saturatedaqueous NaHCO₃ (20 mL), and water (20 mL). The dichloromethane layer wasconcentrated and the resulting residue was purified via flash columnchromatography using a step gradient of 0-5% methanol indichloromethane. The relevant fractions were combined and concentratedto provide a solid intermediate, 0.78 g (88%). ES-MS m/z 446.1 [M+H]⁺,468.3 [M+Na]⁺.

The solid intermediate (450 mg, 1 mmol) and Tripeptide 42 (534 mg, 1.1eq.) were diluted with a 50% solution of TFA in dichloromethane (10 mL),and the resulting reaction was allowed to stir for 2 h at roomtemperature. Toluene (10 mL) was added to the reaction and the reactionmixture was concentrated. The resulting amine intermediate wasazeotropically dried using toluene (3×20 mL) and dried under vacuumovernight.

The resulting amine intermediate was diluted with dichloromethane (2 mL)and to the resulting solution was added triethylamine (557 μL, 4 eq.),followed by DEPC (203 μL, 1.4 eq.). The reaction mixture was allowed tostir for 4 h at room temperature and reaction progress was monitoredusing HPLC. Upon completion of reaction, the reaction mixture wasdiluted with dichloromethane (30 mL) and the dichloromethane layer waswashed sequentially using saturated aqueous NaHCO₃ (20 mL) and saturatedaqueous NaCl (20 mL). The dichloromethane layer was concentrated and theresulting residue was purified using flash column chromatography in astep gradient of 0-5% methanol in dichloromethane. The relevantfractions were combined and concentrated and the resulting residue wasdried using a dichloromethane:hexane (1:1) to provide a white solidintermediate, 0.64 g (84%). ES-MS m/z 757.5 [M+H]⁺.

The white solid intermediate (536 mg, 0.73 mmol) was diluted withmethanol and to the resulting solution was added 10% Pd/C (100 mg). Thereaction was placed under a hydrogen atmosphere and was allowed to stirat atmospheric pressure and room temperature for 2 h. Reaction progresswas monitored by HPLC and was complete in 2 h. The reaction flask waspurged with argon and the reaction mixture was filtered through a pad ofCelite. The Celite pad was subsequently washed with methanol (30 mL) andthe combined filtrates were were concentrated to yield a gray solidintermediate which was used without further purification. Yield=490 mg(91%). ES-MS m/z 731.6 [M+H], 366.6 [M+2H]²⁺/2.

The gray solid intermediate (100 mg, 0.136 mmol), N-Boc-4-aminobenzoicacid (39 mg, 1.2 eq.) and triethylamine (90 μL, 4 eq.) were diluted withdichloromethane (2 mL) and to the resulting solution was added DEPC (28μL, 1.2 eq.). The reaction mixture was allowed to stir at roomtemperature for 2 h, then the reaction mixture was diluted withdichloromethane (30 mL). The dichloromethane layer was sequentiallywashed with saturated aqueous NaHCO₃ (20 mL) and saturated aqueous NaCl(20 mL). The dichloromethane layer was then concentrated and theresulting residue was purified via flash column chromatography using astep gradient of 0-5% in dichlormethane. The relevant fractions werecombined and concentrated and the resulting residue was dried usingdichloromethane:hexane (1:1) to provide a white solid intermediate.ES-MS m/z 950.7 [M+H]⁺.

The white solid intermediate was diluted with a 50% solution of TFA indichloromethane and allowed to stir for 2 h at room temperature. Toluene(10 mL) was added to the reaction and the reaction mixture wasconcentrated. The resulting residue was azeotropically dried usingtoluene (3×15 mL), to provide a yellow oil which was purified usingpreparative HPLC (C₁₈-RP Varian Dynamax column, 5μ, 100 Å, lineargradient of MeCN from 10 to 95% in 0.05 M Triethylammonium carbonatebuffer, pH 7.0, in 30 min at a flow rate of 10 mL/min). The relevantfractions were combined and concentrated and the resulting residue wasazeotropically dried using MeCN (3×20 mL), to provide Compound 56 aswhite solid: 101 mg (87% over 2 steps). ES-MS m/z 850.6 [M+H]⁻, 872.6[M+Na]⁺.

Example 13 Preparation of Compound 57

Compound 49 (100 mg, 0.14 mmol), Compound 27 (160 mg, 0.15 mmol, 1.1eq.), and HOBt (19 mg, 0.14 mmol, 1.0 eq.) were diluted with DMF (2 mL).After 2 min, pyridine (0.5 mL) was added and the reaction mixture wasmonitored using reverse-phase HPLC. Neither Compound 49 nor Compound 27was detected after 24 h. The reaction mixture was concentrated, and theresulting residue was purified using reverse phase preparative-HPLC(Varian Dynamax column 21.4 mm×25 cm, 5μ, 100 Å, using a gradient run ofMeCN and Et₃N—CO₂ (pH 7) at 20 mL/min from 10% to 100% over 40 minfollowed by 100% MeCN for 20 min). The relevant fractions were pooledand concentrated to provide an off-white solid intermediate. ES-MS m/z1608.7 [M+H]⁺

The off-white solid intermediate was diluted with MeCN/water/TFA in an85:5:10 ratio, respectively. The reaction mixture was monitored usingHPLC and was complete in 3 h. The reaction mixture was directlyconcentrated and the resulting residue was purified using reverse phasepreparative-HPLC (Varian Dynamax column 21.4 mm×25 cm, 5μ, 100 Å, usinga gradient run of MeCN and 0.1% TFA at 20 mL/min from 10% to 100% over40 min followed by 100% MeCN for 20 min). The relevant fractions werecombined and concentrated to provide Compound 57 as an off-white powder.Yield: 46 mg (32% overall); ES-MS m/z 1334.8 [M+H]⁺; UV λ_(max) 215, 256nm.

Example 14 Preparation of Compound 58

Compound 49 (1.69 g, 2.35 mmol), Compound 21 (2.6 g, 3.52 mmol, 1.5eq.), and HOBt (64 mg, 0.45 mmol, 0.2 eq.) were diluted with DMF (25mL). After 2 min, pyridine (5 mL) was added and the reaction wasmonitored using reverse-phase HPLC. The reaction was shown to becomplete in 24 h. The reaction mixture was concentrated to provide adark oil, which was diluted with 3 mL of DMF. The DMF solution waspurified using flash column chromatography (silica gel, eluantgradient:100% dichloromethane to 4:1 dichloromethane-methanol). Therelevant fractions were combined and concentrated to provide an oil thatsolidified under high vacuum to provide a mixture of Compound 58 andunreacted Compound 49 as a dirty yellow solid (R_(f) 0.40 in 9:1dichloromethane-methanol). The dirty yellow solid was diluted with DMFand purified using reverse-phase preparative-HPLC (Varian Dynamax C₁₈column 41.4 mm×25 cm, 8 m, 100 Å, using a gradient run of MeCN and 0.1%aqueous TFA at 45 mL/min from 10% to 100% over 40 min followed by 100%MeCN for 20 min) to provide Compound 58 as an amorphous white powder (Rf0.40 in 9:1 dichloromethane-methanol) which was >95% pure by HPLC andwhich contained less than 1% of Compound 49. Yield: 1.78 g (57%); ES-MSm/z 1316.7 [M+H]⁺; UV λ_(max) 215, 248 nm.

Example 15 Preparation of Compound 59

The hydrochloride salt of Compound 51 (11 mg, 15.2 mmol) and Compound 21(11 mg, 15.2 mmol) were diluted with 1-methyl-2-pyrollidinone (1 mL) andto the resulting solution was added diisopropylethylamine (5.3 mL, 30.3mmol, 2.0 eq.). The mixture was allowed to stir under argon atmospherefor 3 d while being monitored using HPLC. After this time, muchunreacted starting material still remained, HOBt (1.0 eq.) was added andthe reaction mixture was allowed to stir for 24 h, after which time nostarting material remained according to HPLC. The reaction mixture wasconcentrated and the resulting residue was purified usingpreparative-HPLC (Varian Dynamax C₁₈ column 21.4 mm×25 cm, 5 m, 100 Å,using a gradient run of MeCN and water at 20 mL/min from 10% to 100%over 30 min followed by 100% MeCN for 20 min). The relevant fractionswere combined and concentrated to provide Compound 59 as a white solid.Yield: 13 mg (67%); ES-MS m/z 1287.2 [M+H], 1304.3 [M+NH₄]⁺; UV λ_(max)215, 248 nm

Example 16 Preparation of Compound 60

Compound 53 (9 mg, 10.8 μmol) and Compound 28 (5.2 mg, 10.8 μmol) werediluted with dichloromethane (1 mL) and to the resulting solution wasadded HATU (6.3 mg, 16.1 μmol, 1.5 eq.), followed by pyridine (1.3 μL,16.1 μmol, 1.5 eq.). The reaction mixture was allowed to stir underargon atmosphere while being monitored using HPLC. The reaction wascomplete after 6 h. The reaction mixture was concentrated and theresulting residue was diluted with DMSO. The DMSO solution was purifiedusing reverse phase preparative-HPLC (Varian Dynamax column 21.4 mm×25cm, 5μ, 100 Å, using a gradient run of MeCN and 0.1% TFA at 20 mL/minfrom 10% to 100% over 40 min followed by 100% MeCN for 20 min) and therelevant fractions were combined and concentrated to provide an anoff-white solid intermediate which was >95% pure according to HPLC.

The off-white solid intermediate was diluted with dichloromethane (2 mL)and the resulting solution was treated with TFA (0.5 mL). The reactionwas monitored using HPLC, and was complete in 2 h. The reaction mixturewas concentrated, and the resulting residue was diluted with DMSO andpurified under the same conditions as described in Example 13. Therelevant fractions were combined and concentrated to provide Compound 60as an off-white powder. Yield: 14.9 mg (90%); ES-MS m/z 1304.6 [M+H]⁺;UV λ_(max) 215, 275 nm.

Example 17 Preparation of Compound 61

The trifluoroacetate salt of Compound 53 (0.37 g, 0.39 mmol, 1.0 eq.)and Compound 18 (0.30 g, 0.58 mmol, 1.5 eq.) were diluted with DMF (5mL, 0.1 M), and to the resulting solution was added pyridine (95 μL, 1.2mmol, 3.0 eq.). HATU (0.23 g, 0.58 mmol, 1.5 eq.) wa then added as asolid and the reaction mixture was allowed to stir under argonatmosphere while being monitored using HPLC. The reaction progressedslowly, and 4 h later, 1.0 eq. of diisopropylethylamine was added.Reaction was complete in 1 h. The reaction mixture was concentrated invacuo and the resulting residue was purified using preparative-HPLC(Varian Dynamax C18 column 41.4 mm×25 cm, 5μ, 100 Å, using a gradientrun of MeCN and 0.1% aqueous TFA at 45 mL/min from 10% to 100% over 40min followed by 100% MeCN for 20 min) to provide a faint pink solidintermediate.

The pink solid intermediate was diluted with DMF (30 mL) and to theresulting solution was added diethylamine (15 mL). Reaction was completeby HPLC in 2 h. The reaction mixture was concentrated and the resultingresidue was washed twice with ether. The solid intermediate was driedunder high vacuum and then used directly in the next step.

The solid intermediate was diluted with DMF (20 mL) and to the resultingsolution was added MC—OSu (0.12 g, 0.39 mmol, 1.0 eq.). After 4 d, thereaction mixture was concentrated to provide an oil which was purifiedusing preparative-HPLC (Varian Dynamax C18 column 41.4 mm×25 cm, 5μ, 100Å, using a gradient run of MeCN and 0.1% aqueous TFA at 45 mL/min from10% to 100% over 40 min followed by 100% MeCN for 20 min). Compound 61was isolated as a white flaky solid. Yield: 0.21 g (38% overall); ES-MSm/z 1285.9 [M+H]+; 13.07.8 [M+Na]+; UV λ_(max) 215, 266 nm.

Example 18 Preparation of Compound 62

Fmoc-val-cit-PAB-OCO-Pnp (19a) (0.65 g, 0.85 mmol, 1.1 eq.), Compound 49(0.55 g, 0.77 mmol, 1.0 eq.), and HOBt (21 mg, 0.15 mmol, 2.0 eq.) werediluted with DMF (2.0 mL) and dissolved using sonication. To theresulting solution was added pyridine (0.5 mL) and the reaction wasmonitored using HPLC. After 24 h, diisopropylethylamine (1.0 eq.) wasadded and the reaction was allowed to stand without stirring for 24 h.The reaction mixture was concentrated to provide an oil residue. The oilresidue was purified using reverse phase preparative-HPLC (VarianDynamax column 41.4 mm×25 cm, 5μ, 100 Å, using a gradient run of MeCNand 0.1% TFA at 45 mL/min from 10% to 100% over 40 min followed by 100%MeCN for 20 min.) The desired fractions were pooled and concentrated toyield an oil that was precipitated with ether to provide an off-whitesolid intermediate. Yield: 0.77 g (74%); ES-MS m/z 1345.7 [M+H]⁺; UVλ_(max) 215, 254 nm.

The off-white solid intermediate (about 85 mg) was deprotected usingdiethylamine (1 mL) in DMF (3 mL). After 1 h, the reaction was complete.The reaction mixture was concentrated, and the resulting residue wasprecipitated in 1 mL of EtOAc followed by addition of excess ether(about 20 mL). The amine intermediate was filtered and dried under highvacuum and used in the next step without further purification.

The amine intermediate (70 mg, 61 μmol, 1.0 eq.) was taken up in DMF (10mL), and to the resulting solution was added sequentially,bromoacetamidocaproic acid (17 mg, 67 μmol, 1.1. eq.), PyBrop (32 mg, 67μmol, 1.1 eq.), and diisopropylethylamine (16 μL, 92 μmol, 1.5 eq.).After 24 h, an additional 1.0 eq. of bromoacetamidocaproic acid wasadded. Reaction was stopped after 30 h. The reaction mixture wasconcentrated to an oil and the oil purified using reverse phasepreparative-HPLC (Synergi MaxRP C₁₂ column 21.4 mm×25 cm, 5μ, 80 Å,using a gradient run of MeCN and 0.1% TFA at 20 mL/min from 10% to 100%over 40 min followed by 100% MeCN for 20 min.). The relevant fractionswere combined and concentrated to provide Compound 62 as a white solid.Yield: 23 mg (27%); ES-MS m/z 1356.7 [M+H]⁺; UV λ_(max) 215, 247 nm.

Example 19 Preparation of Compound 63

Fmoc-val-cit-PAB-OC(O)-Me-val-val-dil-dap-nor (about 48 mg, obtainedaccording to Example 18) was subjected to Fmoc-removal by treating withdiethylamine (1 mL) in DMF (3 mL). After 1 h, the reaction was complete.The reaction mixture was concentrated and the resulting residue wasprecipitated using 1 mL of EtOAc followed by addition of excess ether(about 20 mL). The amine intermediate was filtered and dried under highvacuum and used in the next step without further purification.

The amine intermediate (35 μmol, 1.1 eq.) was diluted with DMF (2 mL),and to the resulting solution was added sequentially maleimido-PEG acid(Frisch, B.; Boeckler, C.; Schuber, F. Bioconjugate Chem. 1996, 7,180-6; 7.8 mg, 32 μmol, 1.0 eq.), DEPC (10.7 μL, 64 μmol, 2.0 eq.), anddiisopropylethylamine (11.3 μL, 64 μmol, 2.0 eq.). The reaction wascomplete in 15 min according to HPLC. The reaction mixture wasconcentrated to provide an oil. The oil was diluted with 1 mL of DMSOand purified using reverse phase preparative-HPLC (Synergi MaxRP C₁₂column 21.4 mm×25 cm, 5μ, 80 Å, using a gradient run of MeCN and 0.1%TFA at 20 mL/min from 10% to 100% over 40 min followed by 100% MeCN for20 min). The relevant fractions were combined and concentrated toprovide Compound 63 as a white solid.

Yield: 16.2 mg (34%); ES-MS m/z 1348.6 [M+H]⁺; UV λ_(max) 215, 247 nm.

Examples 20-25 describe the conjugation of the monoclonal antibodiescBR96 and cAC10 to a Drug-Linker Compound. These antibodies wereobtained as described in Bowen, et al., J. Immunol. 1993, 151, 5896; andTrail, et al., Science 1993, 261, 212, respectively.

The number of Drug-Linker moities per Ligand in a Drug-Linker-LigandConjugate varies from conjugation reaction to conjugation reaction, buttypically ranges from about 7 to about 9, particularly when the Ligandis cBR96 or cAC10.

Example 20 Preparation of Compound 64

cBR96 Antibody (24 mg) was reduced using DTT as described in GeneralProcedure L, then the number of thiols per antibody and the antibodyconcentration were determined as described in General Procedure M andGeneral Procedure N, respectively. Result: [Ab]=4.7 mg/mL=29.4 μM;[thiol]=265 μM; SH/Ab=9.0 (Typical SH/Ab range is from about 7.8 toabout 9.5).

-   -   Conjugation:

A solution of PBS/DTPA (2.2 mL) as defined above herein, was added to4.2 mL of reduced antibody and the resulting solution was cooled to 0°C. using an ice bath. In a separate flask, a 130.5 μL stock solution ofCompound 57 (8.4 mM in DMSO, 8.5 mol Compound 57 per mol reducedantibody) was diluted with MeCN (1.48 mL, pre-chilled to 0° C. in an icebath). The MeCN solution of Compound 57 was rapidly added to theantibody solution and the reaction mixture was stirred using a vortexinstrument for 5-10 sec., returned to the ice bath and allowed to stirat 0° C. for 1 hr, after which time 218 μL of a cysteine solution (100mM in PBS/DTPA) was then added to quench the reaction. 60 μL of thequenched reaction mixture was saved as a “qrm” sample.

While the reaction proceeded, three PD10 columns (Sephadex G25,available from Sigma-Aldrich, St. Louis, Mo.) were placed in a cold roomand equilibrated with PBS (which had been pre-cooled to 0° C. using anice bath).

The quenched reaction mixture, which contained Compound 64, wasconcentrated to ≦3 mL by ultracentrifugation using two Ultrafree 4centrifuge filtering devices (30K molecular weight cutoff membrane;Millipore Corp.; Bedford, Mass.; used according to manufacturer'sinstructions) which were pre-cooled to 4° C. in a refrigerator and theconcentrated reaction mixture was eluted through the three pre-chilledPD10 columns using PBS as the eluent (1 mL for each column). The elutedconjugate was collected in a volume of 1.4 mL per column, for a totaleluted volume of 4.2 mL. The eluted Conjugate solution was then filteredusing a sterile 0.2 micron syringe-end filter, 250 μL of Conjugatesolution was set aside for analysis, and the remainder of the Conjugatesolution was frozen in sterile vials.

The concentration of Compound 64, the number of Drug molecules perAntibody, the amount of quenched Drug-Linker and the percent ofaggregates were determined using General Procedures P, N, O and Q,respectively.

-   -   Assay Results:    -   [Compound 64]=3.8 mg/mLg    -   % Aggregate=trace    -   Residual Thiol Titration:Residual thiols=1.7/Ab.        Drug/Ab˜9.0−1.7=7.3    -   Quenched Drug-Linker: undetectable    -   Yield: 4.2 mL, 16 mg, 66%.

Example 21 Preparation of Compounds 65

cAC10 Antibody (24 mg) was reduced using DTT as described in GeneralProcedure L, then the number of thiols per antibody and the antibodyconcentration were determined as described in General Procedure M andGeneral Procedure N, respectively.

-   -   Results: [Ab]=4.9 mg/mL=30.7 μM;        -   [thiol]=283 μM; 9.2 SH/Ab    -   Conjugation:

A solution of PBS/DTPA (2.2 mL) as defined above herein, was added to4.2 mL of reduced antibody and the resulting solution was cooled to 0°C. using an ice bath. In a separate flask, 125 μL of a stock solution ofCompound 57 (8.4 mM in DMSO, 8.5 mol Compound 57 per mol reducedantibody) was diluted with MeCN (1.48 mL, pre-chilled to 0° C. in an icebath). The MeCN solution of Compound 57 was rapidly added to theantibody solution and the reaction mixture was stirred using a vortexinstrument for 5-10 sec., then returned to the ice bath and allowed tostir at 0° C. for 1 hr, after which time 218 μL of a cysteine solution(100 mM in PBS/DTPA) was then added to quench the reaction. 60 μL of thequenched reaction mixture was saved as a “qrm” sample.

While the reaction proceeded, four PD10 columns (Sephadex G25, availablefrom Sigma-Aldrich, St. Louis, Mo.) were placed in a cold room andequilibrated with PBS (which had been pre-cooled to 0° C. using an icebath).

The quenched reaction mixture, which contained Compound 65, wasconcentrated to ≦3 mL by ultracentrifugation using two Ultrafree 4centrifuge filtering devices (30K molecular weight cutoff membrane;Millipore Corp.; Bedford, Mass.; used according to manufacturer'sinstructions) which were pre-cooled to 4° C. in a refrigerator and theconcentrated reaction mixture was eluted through the four pre-chilledPD10 columns using PBS as the eluent (1 mL for each column). The elutedconjugate was collected in a volume of 1.4 mL per column, for a totaleluted volume of 5.6 mL. The eluted Conjugate solution was then filteredusing a sterile 0.2 micron syringe-end filter, 250 μL of Conjugatesolution was set aside for analysis, and the remainder of the Conjugatesolution was frozen in sterile vials.

The concentration of Compound 65, the number of Drug molecules perAntibody, the amount of quenched Drug-Linker and the percent ofaggregates were then determined using General Procedures P, N, O and Q,respectively.

-   -   Assay Results:    -   [Compound 65]=2.8 mg/mL    -   % Aggregate=trace    -   Residual Thiol Titration:Residual thiols=1.6/Ab.        Drug/Ab˜9.2−1.6=7.6    -   Not covalently bound Drug-Linker: undetectable    -   Yield: 5.6 mL, 15.7 mg, 65%.

Example 22 Preparation of Compound 66

cBR96 Antibody (24 mg) was was reduced using DTT as described in GeneralProcedure L, then the number of thiols per antibody and the antibodyconcentration were determined as described in General Procedure M andGeneral Procedure N, respectively.

-   -   Result: [Ab]=3.7 mg/mL=23.1 μM; [thiol]=218 μM; 9.4 SH/Ab    -   Conjugation:

A solution of PBS/DTPA (2.2 mL) as defined above herein, was added to4.2 mL of reduced antibody and the resulting solution was cooled to 0°C. using an ice bath. In a separate flask, 145.5 μL of a stock solutionof Compound 58 (8.3 mM in DMSO, 9.0 mol Compound 58 per mol reducedantibody) was diluted with MeCN (1.48 mL, pre-chilled to 0° C. in an icebath). The MeCN solution of Compound 58 was rapidly added to theantibody solution and the reaction mixture was stirred using a vortexinstrument for 5-10 sec., then returned to the ice bath and allowed tostir at 0° C. for 1 hr, after which time 249 μL of a cysteine solution(100 mM in PBS/DTPA) was then added to quench the reaction. 60 μL of thequenched reaction mixture was saved as a “qrm” sample.

While the reaction proceeded, three PD10 columns (Sephadex G25,available from Sigma-Aldrich, St. Louis, Mo.) were placed in a cold roomand equilibrated with PBS (which had been pre-cooled to 0° C. using anice bath).

The quenched reaction mixture, which contained Compound 66, wasconcentrated to ≦3 mL by ultracentrifugation using two Ultrafree 4centrifuge filtering devices (30K molecular weight cutoff membrane;Millipore Corp.; Bedford, Mass.; used according to manufacturer'sinstructions) which were pre-cooled to 4° C. in a refrigerator and theconcentrated reaction mixture was eluted through the three pre-chilledPD10 columns using PBS as the eluent (1 mL for each column). The elutedconjugate was collected in a volume of 1.4 mL per column, for a totaleluted volume of 4.2 mL. The eluted Conjugate solution was then filteredusing a sterile 0.2 micron syringe-end filter, 250 μL of Conjugatesolution was set aside for analysis, and the remainder of the Conjugatesolution was frozen in sterile vials.

The concentration of Compound 66, the number of Drug molecules perAntibody, the amount of quenched Drug-Linker and the percent ofaggregates were determined using General Procedures P, N, O and Q,respectively.

-   -   Assay Results:    -   [Compound 66]=3.0 mg/mL    -   % Aggregate=trace    -   Residual Thiol Titration:Residual thiols=0.4/Ab.        Drug/Ab˜9.5−0.4=9.1    -   Not covalently bound Drug-Linker: 0.3% of 57-Cys adduct    -   Yield: 5.3 mL, 15.9 mg, 66%.

Example 23 Preparation of Compound 67

cAC10 Antibody (24 mg) was reduced using DTT as described in GeneralProcedure L, then the number of thiols per antibody and the antibodyconcentration were determined as described in General Procedure M andGeneral Procedure N, respectively.

-   -   Result: [Ab]=3.9 mg/mL=24.5 μM; [thiol]=227 μM; 9.3 SH/Ab    -   Conjugation:

A solution of PBS/DTPA (2.2 mL) as defined above herein, was added to4.2 mL of reduced antibody and the resulting solution was cooled to 0°C. using an ice bath. In a separate flask, 154.4 μL of a stock solutionof Compound 58 (8.3 mM in DMSO, 9.0 mol Compound 58 per mol reducedantibody) was diluted with MeCN (1.46 mL, pre-chilled to 0° C. in an icebath). The MeCN solution of Compound 58 was rapidly added to theantibody solution and the reaction mixture was stirred using a vortexinstrument for 5-10 sec., then returned to the ice bath and allowed tostir at 0° C. for 1 hr, after which time 249 μL of a cysteine solution(100 mM in PBS/DTPA) was then added to quench the reaction. 60 μL of thequenched reaction mixture was saved as a “qrm” sample.

While the reaction proceeded, four PD10 columns (Sephadex G25, availablefrom Sigma-Aldrich, St. Louis, Mo.) were placed in a cold room andequilibrated with PBS (which had been pre-cooled to 0° C. using an icebath).

The quenched reaction mixture, which contained Compound 67, wasconcentrated to ≦3 mL by ultracentrifugation using two Ultrafree 4centrifuge filtering devices (30K molecular weight cutoff membrane;Millipore Corp.; Bedford, Mass.; used according to manufacturer'sinstructions) which were pre-cooled to 4° C. in a refrigerator and theconcentrated reaction mixture was eluted through the four pre-chilledPD10 columns using PBS as the eluent (1 mL for each column). The elutedconjugate was collected in a volume of 1.4 mL per column, for a totaleluted volume of 5.6 mL. The eluted Conjugate solution was then filteredusing a sterile 0.2 micron syringe-end filter, 250 μL of Conjugatesolution was set aside for analysis, and the remainder of the Conjugatesolution was frozen in sterile vials.

The concentration of Compound 67, the number of Drug molecules perAntibody, the amount of quenched Drug-Linker and the percent ofaggregates were determined using General Procedures P, N, O and Q,respectively.

-   -   Assay Results:    -   [Compound 67]=3.0 mg/mL    -   % Aggregate=trace    -   Residual Thiol Titration:Residual thiols=0.5/Ab.        Drug/Ab˜9.5−0.5=9.0    -   Quenched Drug-Linker: 1.1% of 58-Cys adduct    -   Yield: 5.3 mL, 15.9 mg, 66%.

Example 24 Preparation of Compound 68

cBR96 Antibody (24 mg) was reduced using DTT as described in GeneralProcedure L, then the number of thiols per antibody and the antibodyconcentration were determined as described in General Procedure M andGeneral Procedure N, repectively.

-   -   Result: [Ab]=4.4 mg/mL=27.2 μM; [thiol]=277 μM; 10.2 SH/Ab    -   Conjugation:

The reduced antibody was diluted with DMSO (1.47 mL, pre-chilled to 0°C. in an ice bath) so that the resulting solution was 20% DMSO. Thesolution was allowed to stir for 10 min. at 0° C., then 127.8 μL of astock solution of Compound 60 (7.6 mM solution in DMSO; 9 mol Compound60 per mol antibody) was rapidly added. The reaction mixture wasimmediately stirred using a vortex instrument and return to the ice bathand allowed to stir at 0° C. for 1 hr, after which time 213 μL, of acysteine solution (100 mM in PBS/DTPA) was then added to quench thereaction. 60 μL of the quenched reaction mixture was saved as a “qrm”sample.

While the reaction proceeded, four PD10 columns (Sephadex G25, availablefrom Sigma-Aldrich, St. Louis, Mo.) were placed in a cold room andequilibrated with PBS (which had been pre-cooled to 0° C. using an icebath).

The quenched reaction mixture, which contained Compound 68, wasconcentrated to ≦3 mL by ultracentrifugation using two Ultrafree 4centrifuge filtering devices (30K molecular weight cutoff membrane;Millipore Corp.; Bedford, Mass.; used according to manufacturer'sinstructions) which were pre-cooled to 4° C. in a refrigerator and theconcentrated reaction mixture was eluted through the four pre-chilledPD10 columns using PBS as the eluent (1 mL for each column). The elutedconjugate was collected in a volume of 1.4 mL per column, for a totaleluted volume of 5.6 mL. The eluted Conjugate solution was then filteredusing a sterile 0.2 micron syringe-end filter, 250 μL of Conjugatesolution was set aside for analysis, and the remainder of the Conjugatesolution was frozen in sterile vials.

The concentration of Compound 68, the number of Drug molecules perAntibody, the amount of quenched Drug-Linker and the percent ofaggregates were determined using General Procedures P, N, O and Q,respectively.

Because the absorbances of Compound 60 and antibody largely overlap,spectrophotometric determination of the conjugate concentration requiresthe measurement of absorbance at 270 and 280 nm. The molar concentrationof conjugate is given by the following formula:

[Conjugate]=(OD₂₈₀×1.08e ⁻⁵−OD₂₇₀×8.20e ⁻⁶)×dilution factor,

-   -   where the values 1.08e⁻⁵ and 8.20e⁻⁶ are calculated from the        molar extinction coefficients of the drug and the antibody,        which are estimated as:    -   ε₂₇₀ Compound 60=2.06e4 ε₂₇₀ cBR96=1.87e5    -   ε₂₈₀ Compound 60=1.57e4 ε₂₈₀ cBR96=2.24e5    -   Assay Results:    -   [Compound 68]=3.2 mg/mL    -   % Aggregate=trace    -   Residual Thiol Titration:Residual thiols=1.0/Ab.        Drug/Ab˜10.2−1.0=9.2    -   Quenched Drug-Linker: trace    -   Yield: 5.6 mL, 17.9 mg, 75%.

Example 25 Preparation of Compound 69

cAC10 Antibody (24 mg) was reduced using DTT as described in GeneralProcedure L, then the number of thiols per antibody and the antibodyconcentration were determined as described in General Procedure M andGeneral Procedure N, repectively.

-   -   Result: [Ab]=4.8 mg/mL=29.8 μM; [thiol]=281 μM; 9.4 SH/Ab    -   Conjugation:

The reduced antibody was diluted with DMSO (1.47 mL, pre-chilled to 0°C. in an ice bath) so that the resulting solution was 20% DMSO. Thesolution was allowed to stir for 10 min. at 0° C., then 140 μL of astock solution of Compound 60 (7.6 mM solution in DMSO; 8.5 mol Compound60 per mol antibody) was rapidly added. The reaction mixture wasimmediately stirred using a vortex instrument and return to the ice bathand allowed to stir for 1 hr at 0° C., after which time 213 μL of acysteine solution (100 mM in PBS/DTPA) was then added to quench thereaction. 60 μL of the quenched reaction mixture was saved as a “qrm”sample.

While the reaction proceeded, four PD10 columns (Sephadex G25, availablefrom Sigma-Aldrich, St. Louis, Mo.) were placed in a cold room andequilibrated with PBS (which had been pre-cooled to 0° C. using an icebath).

The quenched reaction mixture, which contained Compound 69, wasconcentrated to ≦3 mL by ultracentrifugation using two Ultrafree 4centrifuge filtering devices (30K molecular weight cutoff membrane;Millipore Corp.; Bedford, Mass.; used according to manufacturer'sinstructions) which were pre-cooled to 4° C. in a refrigerator and theconcentrated reaction mixture was eluted through the four pre-chilledPD10 columns using PBS as the eluent (1 mL for each column). The elutedconjugate was collected in a volume of 1.4 mL per column, for a totaleluted volume of 5.6 mL. The eluted Conjugate solution was then filteredusing a sterile 0.2 micron syringe-end filter, 250 μL of Conjugatesolution was set aside for analysis, and the remainder of the Conjugatesolution was frozen in sterile vials.

The concentration of Compound 69, the number of Drug molecules perAntibody, the amount of quenched Drug-Linker and the percent ofaggregates were determined using General Procedures P, N, O and Q,respectively.

Because the absorbances of Compound 60 and antibody largely overlap,spectrophotometric determination of the conjugate concentration requiresthe measurement of absorbance at 270 and 280 nm. The molar concentrationof conjugate is given by the following formula:

[Conjugate]=(OD₂₈₀×1.08e ⁻⁵−OD₂₇₀×8.20e ⁻⁶)×dilution factor,

-   -   where the values 1.08e⁻⁵ and 8.20e⁻⁶ are calculated from the        molar extinction coefficients of the drug and the antibody,        which are estimated as:    -   ε₂₇₀ Compound 60=2.06e⁴ ε₂₇₀ cAC10=2.10e⁵    -   δ₂₈₀ Compound 60=1.57e⁴ ε₂₈₀ cAC10=2.53e⁵    -   Assay Results:    -   [Compound 69]=3.0 mg/mL    -   % Aggregate=trace    -   Residual Thiol Titration:Residual thiols=0.7/Ab.        Drug/Ab˜9.4−0.7=8.7    -   Quenched Drug-Linker: trace    -   Yield: 5.6 mL, 16.8 mg, 70%.

Example 26 Preparation of Compound 75

Diethyl (4-nitrobenzyl)phosphonate (1.1 g, 4.02 mmol) was diluted inanhydrous THF (4 mL) and the resulting mixture was cooled to 0° C.Sodium hydride (0.17 g, 4.22 mmol, 1.05 eq., 60% dispersion in mineraloil) was added and the resulting reaction was allowed to stir for 5 min.At this time gas evolution from the reaction mixture had ceased.2,2-Dimethyl-1,3-dioxan-5-one (0.52 g, 4.02 mmol) in 1 mL of anydrousTHF was then added to the reaction mixture via syringe and the reactionwas allowed to warm to room temperature with stirring. Additional THF (1mL) was added after 30 min to help dilute the resulting precipitate andthe resulting mixture was stirred for an additional 30 min., wastransferred to a separatory funnel containing EtOAc (10 mL) and water(10 mL). The organic phase was collected, washed with brine, and thecombined aqueous extracts were washed with ethyl acetate (2×). Thecombined organic extracts were dried over MgSO₄, filtered, andconcentrated to provide a dark red crude oil that was purified usingflash chromatography on a silica gel column (300×25 mm) and eluting with9:1 hexanes-EtOAc to provide a pale yellow solid intermediate. Yield:0.57 g (57%); R_(f) 0.24 (9:1 hexanes-EtOAc); UV λ_(max) 225, 320 nm. ¹HNMR (CDCl₃) δ 8.19 (2H, d, J=8.4 Hz), 7.24 (2H, d, J=8.4 Hz), 6.33 (1H,s), 4.62 (2H, s), 4.42 (2H, s), 1.45 (6H, s). ¹³C NMR (CDCl₃) δ 146.6,142.7, 141.3, 129.4, 123.9, 121.1, 99.9, 64.4, 60.8, 24.1.

The pale yellow solid intermediate (0.25 g, 1.0 mmol) was diluted usingTHF (20 mL), the resulting mixture was treated with 1 N HCl (10 mL) andallowed to stir for 5 min. To the reaction mixture was added diethylether (150 mL) and water and the resulting mixture was transferred to aseparatory funnel. The organic layer was dried (MgSO₄), filtered andconcentrated to give an oil. The resulting diol was then taken up inTHF-methanol (1:1, 4 mL each, 0.3 M) followed by the addition of RaneyNickel (100 μL, 100 μL/mmol nitro-group, 50% slurry in water) andhydrazine (74 μL, 1.5 eq.). Gas evolution occurred while the reactionmixture was heated to 50-60° C. After 30 min and 1 h, 1.5 eq. ofhydrazine was added each time. The yellow mixture was filtered throughcelite and washed with methanol. The filtrated was concentrated toprovide Compound 75 as an oil which later crystallized to a yellowsolid. Yield: 0.14 g (78%); UV λmax 215, 260 nm. 1H NMR (DMSO) δ 7.00(2H, d, J=8.4 Hz), 6.51 (2H, d, J=8.4 Hz), 6.33 (1H, s), 5.20 (2H, bs),4.64 (2H, bd), 4.04 (2H, s). 13C NMR (DMSO) δ 147.2, 138.1, 129.6,126.1, 124.6, 113.7, 63.6, 57.5.

Example 27 Preparation of Compound 79

To a mixture of Compound 75 (BHMS, 0.12 g, 0.67 mmol) inmethanol-dichloromethane (1:2, 4.5 mL total) was added Fmoc-Val-Cit(0.33 g, 0.67 mmol) followed by EEDQ (0.25 g, 1.0 mmol, 1.5 eq.) and theresulting reaction was allowed to stir for 15 hours under inertatmosphere. Additional EEDQ (1.5 eq.) and Fmoc-Val-Cit (1.0 eq.) werethen added due to the presence of unreacted BHMS and the resultingreaction was allowed to stir for 2 days and concentrated. The resultingresidue was triturated using ether to provide a tan solid intermediate.ES-MS m/z 659 [M+H]⁺, 681 [M+Na]⁺; UV λ_(max) 215, 270 nm. 1H NMR (DMSO)δ 10.04 (1H, s), 8.10 (1H, d, J=7.2 Hz), 7.87 (2H, d, J=7.6 Hz), 7.72(2H, t, J=7.6 Hz), 7.55 (2H, d, J=8.4 Hz), 7.37-7.43 (3H, m), 7.30 (2H,t, J=7.2 Hz), 7.24 (2H, d, J=8.4 Hz), 6.47 (1H, s), 5.96 (1H, t, J=5.2Hz), 5.39 (1H, s), 4.83 (1H, t, J=5.2 Hz), 4.78 (1H, t, J=5.2 Hz), 4.40(1H, dd, J=5.2, 8.0 Hz), 4.20-4.30 (3H, m), 4.11 (2H, d, J=4.4 Hz), 4.04(2H, d, J=5.2 Hz), 3.91 (1H, t, J=7.2 Hz), 2.84-3.06 (2H, m), 1.91-2.03(1H, m), 1.29-1.74 (4H, m), 0.86 (3H, d, J=6.8 Hz), 0.84 (3H, J=6.8 Hz).

The tan solid intermediate was diluted with DMF (10 mL) and theresulting mixture was treated with diethylamine (5 mL), allowed to stirfor 1 hour and concentrated to provide a tan solid which was dried underhigh vacuum for 3 days. The tan solid was triturated using EtOAc (10 mL)and further precipitated using ether (80 mL) to provide a crude residuewhich was filtered through a sintered glass funnel and dried in vacuo toafford a light tan intermediate. ES-MS m/z 436 [M+H]⁺, 458 [M+Na]⁺; UVλ_(max) 215, 270 nm.

The light tan intermediate was diluted with DMF (10 mL) and treated with6-maleimidocaproic acid hydroxysuccinimde ester (0.16 g, 0.53 mmol, 1eq.). The reaction was allowed to stir for 18 h, additionaldiisopropylethylamine (1.0 eq) was added followed by additional6-maleimidocaproic acid hydroxysuccinimde ester (0.5 eq.). The resultingreaction was allowed to stir for 4 hours, after which time, HPLCindicated that the starting material had been consumed. The reactionmixture was concentrated to provide a crude residue that was trituratedusing EtOAc (10 mL) and then further precipitated using ether (75 mL).The precipitate was and dried overnight to provide a tan/orange powderedintermediate. Overall yield: 0.42 g (quant.). ES-MS m/z 629 [M+H]⁺, 651[M+Na]⁺; UV λ_(max) 215, 270 nm.

The tan/orange powdered intermediate (0.40 g, 0.64 mmol) was partiallydissolved in DMF (20 mL) and to the resulting mixture was addedbis(4-nitrophenyl) carbonate (0.98 g, 3.2 mmol, 5.0 eq.) anddiisopropylethylamine (0.45 mL, 2.5 mmol, 4.0 eq.). The resultingreaction was allowed to stir for about 4 hours, after which time, HPLCmonitoring indicated that no starting material remained and that thereaction mixture contained 2 products in a 3:2 ratio (the desiredbis-carbonate and the 1,3-dioxan-2-one, respectively). The reactionmixture was concentrated and the resulting residue was triturated usingEtOAc (10 mL), then further precipitated using ether (80 mL) in aone-pot manner. The EtOAc-ether mixture was filtered and the solid wasdried to provide Compound 79 as a tan powder which was used withoutfurther purification.

Example 28 Preparation of Compound 80

Compound 49 (202 mg, 0.22 mmol, 2.0 eq., 80% pure) and Compound 79 (180mg, 0.11 mmol, 1.0 eq., 60% pure) were suspended in dry DMF (2 mL, 0.1M) and to the resulting mixture was added HOBt (3 mg, 22 μmol, 0.2 eq.)followed by pyridine (400 μL, ¼ v/v DMF). The resulting reaction wasallowed to stir for 16 h, diluted with DMSO (2 mL) and and the resultingmixture was purified using preparative HPLC (C₁₈-RP column, 5μ, 100 Å,linear gradient of MeCN in water 10 to 100% in 40 min followed by 20 minat 100%, at a flow rate of 50 mL/min) to provide Compound 80 as a whitesolid. Yield: 70 mg (18%). MALDI-TOF MS m/z 2138.9 [M+Na]⁺, 2154.9[M+K]⁺.

Example 29 Preparation of Compound 81

Compound 81 was made using the method described in Example 1 andsubstituting Fmoc-(D)-val-(L)-cit-PAB-OH for Compound 19.

Example 30 Preparation of Compound 82

Compound 82 was made using the method described in Example 1 andsubstituting Fmoc-(L)-val-(D)-cit-PAB-OH for Compound 19.

Example 31 Preparation of Compound 83

Compound 83 was made using the method described in Example 1 andsubstituting Fmoc-(D)-val-(D)-cit-PAB-OH for Compound 19.

Example 32 Preparation of Compound 84

Compound 84 was made using the method described in Example 14 andsubstituting Compound 81 for Compound 21.

Example 33 Preparation of Compound 85

Compound 85 was made using the method described in Example 14 andsubstituting Compound 82 for Compound 21.

Example 34 Preparation of Compound 86

Compound 86 was made using the method described in Example 14 andsubstituting Compound 83 for Compound 21.

Example 35 Preparation of Compound 87

A mixture of 6-Maleimidocaproic acid (1.00 g, 4.52 mmol, 1.0 eq.),p-aminobenzyl alcohol (1.11 g, 9.04 mmol, 2.0 eq.) and EEDQ (2.24 g,9.04 mmol, 2.0 eq.) were diluted in dichloromethane (13 mL). Theresulting reaction was stirred about 16 hr., then concentrated andpurified using flash column chromatography in a step gradient 25-100%EtOAc in hexanes to provide a solid intermediate. Yield: 1.38 g (96%);ES-MS m/z 317.22 [M+H]⁺, 339.13 [M+Na]⁺; UV λ_(max) 215, 246 nm.

The solid intermediate (0.85 g, 2.69 mmol, 1.0 eq.) andbis(4-nitrophenyl)carbonate (2.45g, 8.06 mmol, 3.0 eq.) were diluted inDMF (10 mL), and to the resulting mixture was addeddiisopropylethylamine (0.94 mL, 5.37 mmol, 2.0 eq.). The resultingreaction was stirred for about 1 hr, after which time RP-HPLC indicatedthat the reaction was complete. The reaction mixture was concentrated invacuo, and the resulting crude residue was triturated using diethylether (about 250 mL) to provide a white solid intermediate uponfiltration. Yield: 1.25 g (96%); UV λ_(max) 215, 252 nm.

The white solid intermediate (259 mg, 0.0538 mmol, 1.0 eq.), MMAE (464mg, 0.646 mmol, 1.2 eq.), and HOBt (14.5 mg, 0.108 mmol, 0.2 eq.) werediluted in pyridine/DMF (1:5, 6 mL), and the resulting reaction wasstirred for about 10 h, after which time RP-HPLC indicated incompletereaction. The reaction mixture was concentrated, the resulting cruderesidue was diluted using DMF (3 mL), and to the resulting mixture wasadded diisopropylethylamine (0.469 mL, 0.538 mmol, 1.0 eq.) and theresulting reaction was allowed to stir for about 16 hr. The reactionmixture was directly purified using Chromatotron® (radial thin-layerchromatography) with a step gradient (0-5% methanol in dichloromethane),to provide Compound 87 as a white solid. Yield: 217 mg (38%); ES-MS m/z1082.64 [M+Na]⁺; UV λ_(max) 215, 248 nm.

Example 36 Preparation of Compound 88

Fmoc-val-cit (U.S. Pat. No. 6,214,345 to Firestone et al.) was suspendedin dichloromethane (50 mL) and the resulting mixture was treated with33% HBr in HOAc (20 mL), which was added via pipette over about 5minutes. After stirring for about 10 minutes, the reaction mixture wasshown to be complete using HPLC. The reaction mixture was diluted withice (about 500 mL) and saturated aqueous sodium bicarbonate was slowlyadded while stirring until gas evolution ceased. The resultinggelatinous mass was filtered and washed with distilled water to providea solid which was dried under high vacuum in the presence of P₂O₅ for 24h. The resulting tan powdered intermediate (Fmoc-val-cit-PAB-Br) wasabout 70% pure by HPLC and was used without further purification.

The tan powdered intermediate (30 mg, 40.6 μmol) and Compound 53 (34 mg,40.6 μmol) were dissolved in DMF (1 mL), and to the resulting mixturewas added diisopropylethylamine (21 μL, 0.12 mmol, 3.0 eq.). Theresulting reaction was allowed to stir for 6 h, diluted with DMSO (1 mL)and immediately purified using preparative-HPLC (C₁₂-RP column, 5μ, 100Å, linear gradient of MeCN in water (containing 0.1% formic acid) 10 to100% in 40 min followed by 20 min at 100%, at a flow rate of 25 mL/min),to provide as a slight tan powdered intermediate. Yield: 5 mg (8%);ES-MS m/z 1420 [M+H]⁺, 1443 [M+Na]⁺; UV λ_(max) 205, 258 nm.

The slight tan powdered intermediate (4 mg, 9.51 μmol) was diluted usingDMF (1 mL) and the resulting mixture was treated with diethylamine (0.5mL). The resulting reaction was complete in 1 h according to HPLC. Thereaction mixture was concentrated to provide an oily solid residue whichwas triturated with ether (3×) to provide a crude residue. The cruderesidue was diluted with DMF (1 mL) and to the resulting mixture wasadded 6-maleimidocaproic acid hydroxysuccinimide ester (3 mg, 9.5 mop.The resulting reaction was allowed to stir at room temperature for about16 h. The reaction mixture was directly purified using preparative-HPLC(C₁₂-RP column, 5μ, 100 Å, linear gradient of MeCN in water (containing0.1% formic acid) 10 to 100% in 40 min followed by 20 min at 100%, at aflow rate of 25 mL/min) to provide Compound 88 as a slight tan solid.Yield: 3.9 mg (quant); ES-MS m/z 1391 [M+H]⁺; UV λ_(max) 205, 250 nm.

Example 37 Preparation of Compound 89

Preparation of Compound 89A

Compound 89A was prepared using the method described in Example 9 andsubstituting tripeptide Compound 43 for tripeptide Compound 42,intermediate

Preparation of Compound 89

Compound 89a (0.13 g, 0.15 μmol, 1.0 mmol), Compound 21 (0.12 g, 0.17mmol, 1.1 eq.), and HOBt (4 mg, 31 μmol, 0.2 eq.) were suspended inDMF/pyridine (2 mL/0.5 mL, respectively). The resulting reaction wasallowed to stir for about 4 h, then diisopropylethylamine (27 μL, 0.15mmol, 1.0 eq.) was added and the resulting reaction was allowed tostirred for about 54 h and concentrated in vacuo. The resulting crudeoil was diluted with DMSO and purified using preparative-HPLC (C₁₂-RPcolumn, 5μ, 100 Å, linear gradient of MeCN in water (containing 0.1%TFA) 10 to 100% in 40 min followed by 20 min at 100%, at a flow rate of25 mL/min) to provide to a yellow oil that was taken up in a minimumamount of dichloromethane and precipitated with excess ether to affordCompound 89 as a tan powder. Yield: 0.15 mg (68%). ES-MS m/z 1449.14[M+H]⁺; UV λ_(max) 215, 258 nm.

Example 38 Preparation of Compound 90

1,4-Phenylenediamine dihydrochloride (3.06 g, 17 mmoles) and di-t-butyldicarbonate (3.69 g, 17 mmoles) were diluted with 30 mL ofdichloromethane. To the resulting mixture was addeddiisopropylethylamine (8.83 ml, 50.7 mmoles, 3.0 eq.) and the resultingreaction was allowed to stirred for 1 hr. The reaction mixture wastransferred to a seperatory funnel and the organic phase was washedwater (3×10 ml). The organic layer was stored at 4° C. for about 15 hand and crystalization of the product occurred. The crystals werecollected by filtration and washed with cold dichloromethane to provideCompound 90 as a crystalline solid. (1.2 g, 34%). UV λ_(max) 215, 250nm. ¹H NMR (DMSO) δ 8.78 (1H, bs), 7.04 (2H, bd, J=7.2 Hz), 6.43 (2H, d,J=7.2 Hz), 4.72 (2H, s), 1.41 (9H, s).

Example 39 Preparation of Compound 91

A solution of cAC10 (10 mg/mL in 25 mM sodium citrate, 250 mM sodiumchloride, 0.02% Tween 80, pH 6.5) was adjusted to pH 7.5 by addition of0.3 M sodium phosphate, dibasic. To this pH-adjusted cAC10 solution,EDTA was added to a final concentration of 5 mM. The cAC10 solution wasthen pre-heated to 37° C. by incubation in a temperature-controlledoven. After the temperature of the cAC10 solution has equilibrated to37° C., DTT (from a stock solution of 10 mM) was added to achieve afinal DTT-to-cAC 10 molar ratio of about 3.0 in the reduction reaction(a molecular weight of 148,500 Da was used for cAC10). The reductionreaction was then allowed to proceed for 2 hours at 37° C.

At the end of the incubation, the reduction reaction was cooled to aninternal temperature of 2 to 8° C. in an ice-water bath. The temperatureof the solution was kept at 2 to 8° C. throughout the remainingconjugation steps. The chilled reduction reaction was subjected toconstant-volume diafiltration to remove excess DTT using a 30 kDamembrane and the buffer was exchanged into phosphate buffered saline, pH7.4 (PBS). Following diafiltration, the concentration of free thiol inthe reduced and diafiltered cAC10 was determined using General ProcedureM. Conjugation is then carried out by addition of a 15% molar excess ofCompound 58 (from a stock solution of 13 mg/mL in DMSO) relative to thetotal thiols determined using General Procedure M. Additional DMSO wasadded to the conjugation reaction to achieve a final DMSO concentrationof 15% (v/v). The conjugation reaction was allowed to proceed for atotal of 30 min.

At the end of the conjugation reaction, any unreacted excess Drug-Linkercompound was quenched by addition of excess Cysteine (2× molar excessrelative to the total thiols determined using General Procedure M,performed on the reduced and diafiltered cAC10 to produce the quenchedreaction mixture. The quenched reaction mixture is then purified free ofsmall-molecule contaminants via constant-volume diafiltration using a 30kDa membrane and the buffer was exchanged into PBS, pH 7.4. Afterdiafiltration, the conjugate was sterile-filtered using a 0.22 micronfilter to provide Compound 91 in a clear, colorless solution.

Example 40 Preparation of Compound 92

Compound 92 was prepared using the method described in Example 39 usingan amount of DTT (from a stock solution of 10 mM) which provides a finalDTT-to-cAC10 molar ratio of about 1.5 in the reduction reaction.

In vitro Cytotoxicity Experiments

The cell lines used were H3396 human breast carcinoma (cBR96 antigenpositive, cAC10 antigen negative), HCT-116 human colorectal carcinoma(cBR96 and cAC10 antigen negative), and Karpas human anaplastic largecell lymphoma (ALCL) (cBR96 antigen negative, cAC10 antigen positive).These cell lines are available from ATCC. CD30-positive Hodgkin'sDisease (HD) cell line L540 and the ALCL cell line Karpas 299 wereobtained from the Deutsche Sammlung von Mikroorganism and ZellkulturenGmbH (Braunschweig, Germany). L540cy, a derivative of the HD line L540adapted to xenograft growth, was provided by Dr. Phil Thorpe (U of TexasSouthwestern Medical Center, Dallas, Tex.). Cell lines were grown inRPMI-1640 media (Life Technologies Inc., Gaithersburg, Md.) supplementedwith 10% fetal bovine serum.H3396 cells in RPMI containing 10% fetalbovine serum (referred to as medium) were plated in 96-well plates atapproximately 3,000-10,000 cells/well and allowed to adhere overnight.The non-adherent Karpas cell line was plated out at approximately 10,000cells/well at the initiation of the assay. Various concentrations ofillustrative Compounds of the Invention in medium were added intriplicate, and after the times indicated IN FIGS. 1-7, the medium wasremoved, and the cells were washed with fresh medium three times. Aftera 96 hour incubation period at 37° C., Alamar Blue was added and cellviability was determined 4 hours later as described by Ahmed S A, GogalR M Jr, Walsh J E., J. Immunol. Methods, 170, 211-224, 1994.

C.B.-17 SCID (Harlan, Indianapolis, Ind.) mice were used for in vivoexperiments

Example 41 In Vitro Cytotoxicity Data

The cytotoxic effects of Compound 49 and Compound 53 on H3396 humanbreast carcinoma cells are shown in FIG. 1. The data show that afterexposure for 1 hour, Compound 53 is more cytotoxic than Compound 49 atconcentrations of up to 0.01 mM. The compounds show substantially equalcytotoxicity at concentrations between 0.01 mM and 1.0 mM.

Example 42 In Vitro Cytotoxicity Data

FIG. 2 shows the cytotoxic effects of Compounds 64, 65, 68 and 69 onH3396 human breast carcinoma cells (cBR96 antigen positive, cAC10antigen negative). The data show that the Compounds 64 and 68demonstrate similar and significant cytotoxicity, while Compounds 65 and69 are less efficacious, but nevertheless cytotoxic against H3396 cellsin this particular assay.

Example 43 In Vitro Cytotoxicity Data

FIG. 3 shows the cytotoxic effects of Compounds 64, 65, 68 and 69 onHCT-116 human colorectal carcinoma cells (cBR96 antigen negative, cAC10antigen negative). The data illustrate that none of Compounds 64, 65, 68and 69 is cytotoxic toward the antigen negative HCT-116 cells in thisassay.

Example 44 In Vitro Cytotoxicity Data

FIG. 4 illustrates the cytotoxicity of Compounds 66 and 68 on H3396human breast carcinoma cells (cBR96 antigen positive). The data showthat both Compounds are highly cytotoxic at concentrations above 0.1 mMand that Compound 68 demonstrates greater cytotoxicity than Compound 66at concentrations between 0.01 mg/mL and 0.4 mg/mL.

Example 45 In Vitro Cytotoxicity Data

FIG. 5 illustrates the cytotoxicity of Compounds 66, 68 and 69 on Karpashuman anaplastic large cell lymphoma (cBR96 antigen negative, cAC10antigen positive). The data show that Compound 69 was more cytotoxictoward Karpas cells than compared to Compounds 68 and 66 in this assay.Compound 69 demonstrated significant cytotoxicity at concentrationsabove 0.001 mM, while Compound 66 and Compound 68 were not cytotoxic atconcentrations below 1.0 mg/mL.

Example 46 In Vitro Cytotoxicity Data

FIG. 6 illustrates the cytotoxicity of Compound 66 and 67 at 2h and 96 hon H3396 human breast carcinoma cells (cBR96 antigen positive, cAC10antigen negative). The data show that Compound 66 is highly cytotoxic atconcentrations above 100 mg/mL at short-term exposure (2 h) mg/mL, andat concentrations above 100 mg/mL over long-term exposure (96 h).Compound 67 did not demonstrate cytotoxicity against H3396 cells in thisassay at concentrations up to 1000 mg/mL.

General Procedure S: In Vivo Testing of Selected Drug-Linker-AntibodyConjugates. For the L2987 human adenocarcinoma cell line, Athymic nudemice (8-10 weeks old) were implanted with xenograft tumors or tumorcells. For the Karpas human anaplastic large cell lymphoma model, CB-17SCID mice were implanted subcutaneously with 5×10⁶ cells. In both tumormodels, therapy was initiated once the tumors reached an average volumeof 100 mm³. Groups of mice were injected with one of Compounds 66-69 inphosphate buffered saline intravenously every fours days for a total of6 injections for L2987 animals and 2 injections for Karpas animals.Tumor volume was computed using the formula: 0.5 (longestdimension×perpendicular dimension²). Mice were removed from the studywhen their tumors were approximately 1000 mm³, at which point theaverage tumor sizes from the particular group were no longer plotted.

Example 47 In Vivo Therapeutic Efficacy on 12987 Tumors

FIG. 7 shows the therapeutic effects of Compounds 66-69 on L2987 humanlung adenocarcinoma xenograft tumors (cBR96 antigen positive, cAC10antigen negative) implanted in athymic nude mice. General Procedure Swas followed using subcutaneous

L2987 human lung tumors (from in vivo passaging). Mice were administeredby injection with one of Compounds 66, 67, 68 or 69 at four dayintervals for a total of 6 injections. The first injection was given at15 days post tumor-implant. The data illustrate that administration ofCompound 66 and Compound 68 markedly reduced tumor volume and noadditional growth was noted in treated mice for at approximately 25 daysafter the last injection. Compound 67 and Compound 69 were lessefficacious but nevertheless inhibited tumor cell multiplication in thetreated mice. Testing was stopped in animals receiving Compounds 67 and69 when tumor volume exceeded 1000 mm³.

Example 48 In Vivo Therapeutic Efficacy on Karpas Tumors

FIG. 8 shows the therapeutic effects of compounds 66-69 on Karpas humananaplastic large cell lymphoma xenograft tumors (cAC10 antigen positive,cBR96 antigen negative) implanted in nude mice. General Procedure S wasfollowed using Karpas human anaplastic large cell lymphoma model, CB-17SCID mice were implanted subcutaneously with 5×10⁶ cells. Mice weredosed intravenously with one of Compounds 66, 67, 68 or 69 at four dayintervals for a total of 2 injections starting on day 8. The dataillustrate that Compounds 67 and 69 induced complete regressions, andthat the tumors progressed in animals that received substantiallyequivalent amounts of Compounds 66 and 68.

Example 49 Determination of Cytotoxicity of Selected Compounds in CD30−and CD30+ Cells

Following their physical characterization, the in vitro cytotoxicity ofCompounds 67, 91 and 92 was evaluated in CD30⁺ Karpas 299 and CD30⁻ Rajicells using the Alamar Blue assay as described above. The percent viablecells was plotted versus concentration for each molecule to determinethe IC₅₀ (defined as the mAb concentration that gave 50% cell kill).

Compound 67 demonstrated activity against Karpas 299 cells with an IC₅₀of 4 ng/mL. The IC₅₀ was inversely proportional to drug loading as itincreased from 4 ng/mL for Compound 67 to 7 ng/mL for Compound 91, to 40ng/mL for Compound 92. Selectivity of the tested compounds was evaluatedusing the antigen-negative Raji cell line which were insensitive to allcAC10-containing Compounds with IC₅₀ values >1000 ng/ml for Compounds67, 91 and 92.

Example 50 Cytotoxicity of Selected Compounds in Xenograft Models of HDand ALCL

Cytotoxicity of Compounds 67, 91 and 92 was evaluated in subcutaneousKarpas 299 human anaplastic large cell lymphoma and L540cy Hodgkin'sDisease xenograft models in C.B.-17 SCID mice. Evaluations wereinitiated when tumor volumes averaged 50-100 mm³. Cohorts of Karpas-299bearing mice were injected q4dx4 with Compound 92, Compound 91, orCompound 67 at either 0.25 mg/kg or 0.5 mg/kg. None of the animalstreated at 0.25 mg/kg had a regression, although there was a delay intumor growth compared to untreated controls for the animals treated withCompound 91 and Compound 67. Treatment of Karpas tumors with Compound 91and Compound 67 at 0.5 mg/kg given q4dx4 achieved 5/5 completeregressions and 4/5 complete regressions, respectively. A delay in tumorgrowth compared to untreated animals was observed for Compound 92 at 0.5mg/kg given q4dx4, but no complete regressions were obtained.

Efficacy was also tested in a subcutaneous Karpas model with selectedcompounds administered as a single dose. Compound 91 and Compound 67were injected at single doses of 0.25, 0.5 and 2.0 mg/kg. At the dose of0.25 mg/kg there was no antitumor activity in either group and meantumor volume did not deviate from the untreated controls. A delay in thetumor growth was demonstrated by both molecules at 0.5 mg/kg, but nocomplete regressions were obtained. Treating the mice with Compound 91and Compound 67 at 2 mg/kg achieved 100% complete regressions in bothgroups.

Compound 91 and Compound 67 were also compared in mice bearingsubcutaneous L540cy human HD tumors treated q4dx4 with Compound 91 andCompound 67 at 1 and 3 mg/kg. At 1 mg/kg, mice treated with Compound 91and Compound 67 had significant delays in tumor growth compared to theuntreated animals. Complete regressions were observed in miceadministered with both Compound 91 and Compound 67 at 3 mg/kg.

The present invention is not to be limited in scope by the specificembodiments disclosed in the examples which are intended asillustrations of a few aspects of the invention and any embodiments thatare functionally equivalent are within the scope of this invention.Indeed, various modifications of the invention in addition to thoseshown and described herein will become apparent to those skilled in theare and are intended to fall within the scope of the appended claims.

A number of references have been cited, the entire disclosures of whichare incorporated herein by reference.

1. A compound of the Formula Ia:

or a pharmaceutically acceptable salt thereof, wherein, L- is anantibody that binds to an antigen expressed on an activated humanlymphocyte, wherein the activated human lymphocyte is associated with anautoimmune disease; -A- is a Stretcher unit; a is 1; each —W— isindependently an Amino Acid unit; —Y— is a Spacer unit; w is an integerranging from 2 to 12; y is 1 or 2; p ranges from 1 to about 20; and -Dis a Drug unit of the formula:

the wavy line indicates the point of attachment to the Spacer unit, andindependently at each location: R² is selected from the group consistingof —H and —C₁-C₈ alkyl; R³ is selected from the group consisting of —H,—C₁-C₈ alkyl, —C₃-C₈ carbocycle, —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈ carbocycle), —C₃-C₈ heterocycle and—C₁-C₈ alkyl-(C₃-C₈ heterocycle); R⁴ is selected from the groupconsisting of —H, —C₁-C₈ alkyl, —C₃-C₈ carbocycle, —O—(C₁-C₈ alkyl),-aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈ carbocycle), —C₃-C₈heterocycle and —C₁-C₈ alkyl-(C₃-C₈ heterocycle); R⁵ is selected fromthe group consisting of —H and -methyl; or R⁴ and R⁵ join and form aring with the carbon atom to which they are attached and R⁴ and R⁵ havethe formula —(CR^(a)R^(b))_(n)— wherein R^(a) and R^(b) areindependently selected from the group consisting of —H, —C₁-C₈ alkyl and—C₃-C₈ carbocycle and n is selected from the group consisting of 2, 3,4, 5 and 6; R⁶ is selected from the group consisting of —H and —C₁-C₈alkyl; R⁷ is selected from the group consisting of —H, —C₁-C₈ alkyl,—C₃-C₈ carbocycle, —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈alkyl-(C₃-C₈ carbocycle), —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈heterocycle); each R⁸ is independently selected from the groupconsisting of —H, —OH, —C₁-C₈ alkyl, —C₃-C₈ carbocycle and —O—(C₁-C₈alkyl); R⁹ is selected from the group consisting of —H and —C₁-C₈ alkyl;R¹⁰ is selected from the group consisting of:

Z is —O—, —S—, —NH— or —N(R¹⁴)—; R¹¹ is selected from the groupconsisting of —H, —OH, —NH₂, —NHR¹⁴, —N(R¹⁴)₂, —C₁-C₈ alkyl, —C₃-C₈carbocycle, —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈alkyl-(C₃-C₈ carbocycle), —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈heterocycle); or R¹¹ is an oxygen atom which forms a carbonyl unit (C═O)with the carbon atom to which it is attached and a hydrogen atom on thiscarbon atom is replaced by one of the bonds in the (C═O) double bond;each R¹² is independently selected from the group consisting of -aryland —C₃-C₈ heterocycle; R¹³ is selected from the group consisting of —H,—OH, —NH₂, —NHR¹⁴, —N(R¹⁴)₂, —C₁-C₈ alkyl, —C₃-C₈ carbocycle, —O—(C₁-C₈alkyl), -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈ carbocycle), C₃-C₈heterocycle and —C₁₋₈ alkyl-(C₃-C₈ heterocycle); and each R¹⁴ isindependently —H or —C₁-C₈ alkyl; wherein upon delivery of the compoundof Formula Ia into the activated human lymphocyte, the drug unit isliberated.
 2. A compound of the formula Ia:

or a pharmaceutically acceptable salt thereof, wherein, L- is anantibody that binds to an antigen expressed on an activated humanlymphocyte, wherein the activated human lymphocyte is associated with anautoimmune disease; -A- is a Stretcher unit; a is 1; each —W— isindependently an Amino Acid unit; —Y— is a Spacer unit; w is an integerranging from 2 to 12; y is 1 or 2; p ranges from 1 to about 20; and -Dis a Drug unit having the structure:

the wavy line indicates the point of attachment to the Spacer unit, andindependently at each location: R² is selected from the group consistingof —H and -methyl; R³ is selected from the group consisting of —H,-methyl, and -isopropyl; R⁴ is selected from the group consisting of —Hand -methyl; R⁵ is selected from the group consisting of -isopropyl,-isobutyl, -sec-butyl, -methyl and -t-butyl or R⁴ and R⁵ join and form aring with the carbon atom to which they are attached and R⁴ and R⁵ havethe formula —(CR^(a)R^(b))_(n)— wherein R^(a) and R^(b) areindependently selected from the group consisting of —H, —C₁-C₈ alkyl,and —C₃-C₈ carbocycle, and n is selected from the group consisting of 2,3, 4, 5 and 6; R⁶ is selected from the group consisting of —H and-methyl; each R⁸ is independently selected from the group consisting of—OH, -methoxy and -ethoxy; R¹⁰ is selected from the group consisting of:

R²⁴ is selected from the group consisting of H and —C(O)R²⁵—; R²⁵ isselected from the group consisting of —C₁-C₈ alkyl, —C₃-C₈ carbocycle,-aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈ carbocycle), —C₃-C₈heterocycle and —C₁-C₈ alkyl-(C₃-C₈ heterocycle); Z is —O—, —NH—,—OC(O)—, —NHC(O)—, or —NR²⁸C(O)—; R²⁸ is selected from the groupconsisting of —H and —C₁-C₈ alkyl; n is 0 or 1; and R²⁷ is selected fromthe group consisting of —H, —N₃, —C₁-C₈ alkyl, —C₃-C₈ carbocycle, -aryl,—C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈ carbocycle), —C₃-C₈ heterocycleand —C₁-C₈ alkyl-(C₃-C₈ heterocycle) when n is 0; and R²⁷ is selectedfrom the group consisting of —H, —C₁-C₈ alkyl, —C₃-C₈ carbocycle, -aryl,—C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈ carbocycle), —C₃-C₈ heterocycleand —C₁-C₈ alkyl-(C₃-C₈ heterocycle) when n is 1; wherein upon deliveryof the compound of Formula Ia into the activated human lymphocyte, thedrug unit is liberated.
 3. The compound or a pharmaceutically acceptablesalt of the compound of claim 1 wherein —Y— is a self-immolative spacerunit.
 4. The compound or a pharmaceutically acceptable salt of thecompound of claim 3 wherein R² is —C₁-C₈ alkyl.
 5. The compound or apharmaceutically acceptable salt of the compound of claim 3 wherein R¹⁰is


6. The compound or a pharmaceutically acceptable salt of the compound ofclaim 2 wherein —Y— is a self-immolative spacer unit.
 7. The compound ora pharmaceutically acceptable salt of the compound of claim 6 wherein R²is methyl.
 8. The compound or a pharmaceutically acceptable salt of thecompound of claim 6 wherein R¹⁰ is


9. A compound or a pharmaceutically acceptable salt of the compound ofclaim 3 where -D is a Drug unit having the structure


10. A compound or a pharmaceutically acceptable salt of the compound ofclaim 3 wherein the antibody is bound to the Stretcher unit via a sulfuratom of the antibody.
 11. The compound or a pharmaceutically acceptablesalt of the compound of claim 3 wherein the antibody comprises a humanimmunoglobulin constant region.
 12. The compound or a pharmaceuticallyacceptable salt of the compound of claim 11 wherein the antibodycomprises a IgG1 constant region.
 13. The compound or a pharmaceuticallyacceptable salt of the compound of claim 3 where —W_(w)— is-valine-citrulline-, the amino terminus of —W_(w)— forming a bond with aStretcher unit, and the C-terminus of —W_(w)— forming a bond with aSpacer unit.
 14. The compound or a pharmaceutically acceptable salt ofthe compound of claim 3 wherein p ranges from 1 to about
 5. 15. Thecompound of claim 3 having the structure

or a pharmaceutically acceptable salt thereof, where E is —CH₂— or—CH₂CH₂O—; e is an integer ranging either from 0-10 when E is —CH₂—, orfrom 1-10 when E is —CH₂CH₂—O—; F is —CH₂—; and f is 0 or
 1. 16. Thecompound of claim 3 having the structure

or a pharmaceutically acceptable salt thereof.
 17. A method for treatingan autoimmune disease, comprising administering to an animal in needthereof an effective amount of a compound of Formula Ia:

or a pharmaceutically acceptable salt thereof, wherein, L- is anantibody that binds to an antigen expressed on an activated humanlymphocyte, wherein the activated human lymphocyte is associated with anautoimmune disease; -A- is a Stretcher unit; a is 1; each —W— isindependently an Amino Acid unit; —Y— is a self-immolative Spacer unit;w is an integer ranging from 2 to 12; y is 1 or 2; p ranges from 1 toabout 20; and -D is a Drug unit of the formula:

the wavy line indicates the point of attachment to the Spacer unit, andindependently at each location: R² is —C₁-C₈ alkyl; R³ is selected fromthe group consisting of —H, —C₁-C₈ alkyl, —C₃-C₈ carbocycle, —O—(C₁-C₈alkyl), -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈ carbocycle),—C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈ heterocycle); R⁴ is selectedfrom the group consisting of —H, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,—O—(C₁-C₈ alkyl), -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈carbocycle), —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈ heterocycle); R⁵is selected from the group consisting of —H and -methyl; or R⁴ and R⁵join and form a ring with the carbon atom to which they are attached andR⁴ and R⁵ have the formula —(CR^(a)R^(b))_(n)— wherein R^(a) and R^(b)are independently selected from the group consisting of —H, —C₁-C₈ alkyland —C₃-C₈ carbocycle and n is selected from the group consisting of 2,3, 4, 5 and 6; R⁶ is selected from the group consisting of —H and —C₁-C₈alkyl; R⁷ is selected from the group consisting of —H, —C₁-C₈ alkyl,—C₃-C₈ carbocycle, —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈alkyl-(C₃-C₈ carbocycle), —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈heterocycle); each R⁸ is independently selected from the groupconsisting of —H, —OH, —C₁-C₈ alkyl, —C₃-C₈ carbocycle and —O—(C₁-C₈alkyl); R⁹ is selected from the group consisting of —H and —C₁-C₈ alkyl;R¹⁰ is selected from the group consisting of:

Z is —O—, —S—, —NH— or —N(R¹⁴)—; R¹¹ is selected from the groupconsisting of —H, —OH, —NH₂, —NHR¹⁴, —N(R¹⁴)₂, —C₁-C₈ alkyl, —C₃-C₈carbocycle, —O—(C₁-C₈ alkyl), -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈alkyl-(C₃-C₈ carbocycle), —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈heterocycle); or R¹¹ is an oxygen atom which forms a carbonyl unit (C═O)with the carbon atom to which it is attached and a hydrogen atom on thiscarbon atom is replaced by one of the bonds in the (C═O) double bond;each R¹² is independently selected from the group consisting of -aryland —C₃-C₈ heterocycle; R¹³ is selected from the group consisting of —H,—OH, —NH₂, —NHR¹⁴, —N(R¹⁴)₂, —C₁-C₈ alkyl, —C₃-C₈ carbocycle, —O—(C₁-C₈alkyl), -aryl, -C1-C8 alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈ carbocycle), C₃-C₈heterocycle and —C₁₋₈ alkyl-(C₃-C₈ heterocycle); and each R¹⁴ isindependently —H or —C₁-C₈ alkyl; wherein upon delivery of the compoundof Formula Ia into the activated human lymphocyte, the drug unit isliberated.
 18. A method for treating an autoimmune disease, comprisingadministering to an animal in need thereof an effective amount of acompound of formula Ia:

or a pharmaceutically acceptable salt thereof, wherein, L- is anantibody that binds to an antigen expressed on an activated humanlymphocyte, wherein the activated human lymphocyte is associated with anautoimmune disease; -A- is a Stretcher unit; a is 1; each —W— isindependently an Amino Acid unit; —Y— is a self-immolative Spacer unit;w is an integer ranging from 2 to 12; y is 1 or 2; p ranges from 1 toabout 20; and -D is a Drug unit having the structure:

the wavy line indicates the point of attachment to the Spacer unit, andindependently at each location: R² is -methyl; R³ is selected from thegroup consisting of —H, -methyl, and -isopropyl; R⁴ is selected from thegroup consisting of —H and -methyl; R⁵ is selected from the groupconsisting of -isopropyl, -isobutyl, -sec-butyl, -methyl and -t-butyl orR⁴ and R⁵ join and form a ring with the carbon atom to which they areattached and R⁴ and R⁵ have the formula —(CR^(a)R^(b))_(n)— whereinR^(a) and R^(b) are independently selected from the group consisting of—H, —C₁-C₈ alkyl, and —C₃-C₈ carbocycle, and n is selected from thegroup consisting of 2, 3, 4, 5 and 6; R⁶ is selected from the groupconsisting of —H and -methyl; each R⁸ is independently selected from thegroup consisting of —OH, -methoxy and -ethoxy; R¹⁶ is selected from thegroup consisting of:

R²⁴ is selected from the group consisting of H and —C(O)R²⁵—; R²⁵ isselected from the group consisting of —C₁-C₈ alkyl, —C₃-C₈ carbocycle,-aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈ carbocycle), —C₃-C₈heterocycle and —C₁-C₈ alkyl-(C₃-C₈ heterocycle); Z is —O—, —NH—,—OC(O)—, —NHC(O)—, or —NR²⁸C(O)—; R²⁸ is selected from the groupconsisting of —H and —C₁-C₈ alkyl; n is 0 or 1; and R²⁷ is selected fromthe group consisting of —H, —N₃, —C₁-C₈ alkyl, —C₃-C₈ carbocycle, -aryl,—C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈ carbocycle), —C₃-C₈ heterocycleand —C₁-C₈ alkyl-(C₃-C₈ heterocycle) when n is 0; and R²⁷ is selectedfrom the group consisting of —H, —C₁-C₈ alkyl, —C₃-C₈ carbocycle, -aryl,—C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈ carbocycle), —C₃-C₈ heterocycleand —C₁-C₈ alkyl-(C₃-C₈ heterocycle) when n is 1; wherein upon deliveryof the compound of Formula Ia into the activated human lymphocyte, thedrug unit is liberated.
 19. The method of claim 18 wherein the compoundhas the structure

or a pharmaceutically acceptable salt thereof.
 20. The method of claim19 wherein the antibody immunospecifically binds the CD30 antigen.